CN107428820A

CN107428820A - Humanization tau antibody in Alzheimer's

Info

Publication number: CN107428820A
Application number: CN201580072454.2A
Authority: CN
Inventors: M.诺瓦克; E.孔特塞科瓦; B.科瓦切赫; R.斯克拉巴纳
Original assignee: Axon Neuroscience SE
Current assignee: Axon Neuroscience SE
Priority date: 2014-11-19
Filing date: 2015-11-18
Publication date: 2017-12-01
Anticipated expiration: 2035-11-18
Also published as: EP3786182A1; MX2017006663A; AU2015348012B2; RS61431B1; WO2016079597A8; PT3221349T; JP6864763B2; SI3221349T1; AU2015348012A1; RU2730668C2; RU2017120688A; ES2848376T3; CY1123746T1; JP2020141669A; US11319363B2; US20190169276A1; AU2020227007A1; HK1244495A1; LT3221349T; BR112017010498A2

Abstract

The present invention relates to biochemistry, the diagnosis of molecular biology and Alzheimer's, prevention and treatment field.There is provided herein can distinguish the humanized antibody for people tau of normal (health) to pathology (disease is related) tau.

Description

Humanization tau antibody in Alzheimer's

Sequence table

The application includes the sequence table submitted by EFS-Web with ASCII fromat, and by quoting with its entirety It is incorporated herein.The ASCII copies for being created on November 19th, 2014 are named as 11634.6003.00000_SL.txt, Size is 284,497 bytes.

Invention field

The present invention is biochemistry, the diagnosis of molecular biology and Alzheimer's, prevents and treats field.Herein The humanized antibody for people tau is provided, it can distinguish normal (health) and pathology (disease is related) tau.

Background of invention

Alzheimer's (AD) is a kind of advanced brain structure of destruction, such as relating to those brain knots in memory and cognition The progressive neurodegenerative disorders of structure.The disease causes Cognitive deficiency and memory, study, language to fail and had Meaning and the ability decline of purposeful activity.Need the effective method and composition for treating and preventing AD.

In patch and intracellular and extracellular nerve fibril in brain being present outside neuron in AD histologic characteristics Tangle.Patch is mainly made up of beta amyloid albumen (Α β), and the tau for including pathologic form that tangles, such as pathologic Protein tau Rotamer and its aggregation.Generally acknowledged effect of the Protein tau in AD lesions is proved in numerous researchs.For example, Braak show the neurodegenerative most close associations of AD be exist tau tangle rather than Amyloid plaques (Braak, H., et al., Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol 82: 239-259(1991))。

Tau albumen belongs to intrinsic unordered protein families, is characterised by that rigidity three is not present in their physiological environment Tie up structure (Skrabana et al., 2006).However, tau is truncated and Hyperphosphorylationof can cause from intrinsic disordered state to a variety of The pathologic conversion of soluble and insoluble wrong disordered structure (including conjugate spirals silk (PHF) and other aggregations) (Wischik,C.M.,Novak,M.,Edwards,P.C.,Klug,A.,Tichelaar,W.,Crowther,R.A.(1988) .Structural characterization of the core of the paired helical filament of Alzheimer disease,Proc Natl Acad Sci USA 85,4884-8；Wischik,C.M.,Novak,M..H.C.,Edwards,P.C.,Runswick,M.J.,Jakes,R.,Walker,J.E.,Milstein,C., Roth,M.,Klug,A.(1988).Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease,Proc Natl Acad Sci USA 85, 4506-10；Novak et al.,1993；Skrabana et al.,2006；Zilka,N.,et al.Chaperone-like Antibodies Targeting Misfolded Tau Protein:New Vistas in the Immunotherapy of Neurodegenerative Foldopathies.Journal of Alzheimer’s disease 15(2008)169– 179；Kovacech B,Novak M.(2010).Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer’s disease.Curr Alzheimer Res Dec；7(8):708-16)；Kovacech B,Skrabana R,Novak M. (2010).Transition of tau protein from disordered to misordered in Alzheimer's disease.Neurodegener Dis 7:24-27) these structure changes of cause the toxicity of function to increase, or cause natural egg White physiological function is lost, or both (Zilka et al., 2008；Kovacech B,Novak M.(2010).Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer’s disease.Curr Alzheimer Res Dec；7(8):708-16)； Kovacech B,Skrabana R,Novak M.(2010).Transition of tau protein from disordered to misordered in Alzheimer's disease.Neurodegener Dis 7:24-27).)。

Tau physiological function be mediate tubulin monomer be assembled into form neuronal microtubules network micro-pipe (Buee, L.,Bussiere,T.,Buee-Scherrer,V.,Delacourte,A.,Hof,P.R.(2000).Tau protein isoforms,phosphorylation and role in neurodegenerative disorders.Brain Research.Brain Research Reviews.33,95-130).Tau albumen passes through in the C-terminal part of protein Repeat region combination micro-pipe.Butner KA,Kirschner MW.1991.Tau protein binds to microtubules through a flexible array of distributed weak sites.J Cell Biol 115:717-730；Lee G,Neve RL,Kosik KS.1989.The microtubule binding domain of tau protein.Neuron 2:1615-1624.These repetitive structure domains (R1-R4) are differing from each other, but comprising highly conserved 31-32 amino acid (Taniguchi T, Sumida M, Hiraoka S, Tomoo K, Kakehi T, Minoura K, Sugiyama S,Inaka K,Ishida T,Saito N,Tanaka C 2005(Effects of different anti- tau antibodies on tau fibrillogenesis:RTA-1 and RTA-2 counteract tau aggregation.FEBS Lett 579:1399-1404；Taniguchi S,Suzuki N,Masuda M,Hisanaga S, Iwatsubo T,Goedert M,Hasegawa M.Inhibition of heparin-induced tau filament formation by phenothiazines,polyphenols,and porphyrins.J Biol Chem 280:7614- 7623(2005)).In human brain, exist because in the N-terminal part of Protein tau presence or absence of some amino acid and 3 kinds (R1, R3 and R4) or 4 kinds of (R1-R4) repetitive structure domains at the C-terminal of protein and 6 kinds of unique tau different from each other are same Kind type.Referring also to Fig. 1, it shows that 6 kinds of people's isotypes (are respectively 2N4R, 1N4R, 2N3R, 0N4R, 1N3R in the order of presentation With 0N3R SEQ ID NO.151-156).It is partly overlapping with R1-R2 that tau, which is had pointed out, to induce the most strength of microtubule polymerization Sequence 306-VQIVYK-311 (SEQ ID NO.146) and 274-KVQIINKK-281 areas (SEQ ID NO.144).(von Bergen M,Friedhoff P,Biernat J,Heberle J,Mandelkow EM,Mandelkow E.2000.Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif((306)VQIVYK(311))forming beta structure.Proc Natl Acad Sci U S A 97:5129-5134.) ibid.

In addition, tau pathology and physiological function seem by specific used by by full length protein isotype and its fragment Structure conformation and intrinsic disordered structure influence.For example, Kontsekova etc. describes the conformation of the tau intramoleculars of some truncations (cover residue 297-IKHVPGGGSVQIVYKPVDLSKVTSKCGSL-325 (SEQ ID NO in region:145)), itself and those sections Short Protein tau molecule has significant relationship (WO 2004/007547) on the function that micro-pipe is assembled.

In addition to their physiological action, Protein tau repetitive sequence is considered as participation and forms pathologic Protein tau aggregation And other structures.Therefore, to physiological micro-pipe combination duplicate block mediation activity and pathologic micro-pipe combination duplicate block can be distinguished Needs be present in the Protein tau targeted therapy and diagnostic method for mediating activity.For example, pathologic conjugate spirals silk (PHF) Pronase (pronase) resistance core repeats Protein tau isotype by 3 and 4 micro-pipes for repeating Protein tau isotype combine District's groups are into (Jakes, R., Novak, M., Davison, M., Wischik, C.M. (1991) .Identification of 3- and 4-repeat tau isoforms within the PHF in Alzheimer's disease.EMBO J 10, 2725-2729；Wischik, et al., 1988a；Wischik, et al., 1988b).In addition, Novak etc. shows that PHF length is The protease resistant core of 93-95 amino acid be limited to 3 tandem repetitive sequences (Novak, M., Kabat, J., Wischik, C.M.(1993).Molecular characterization of the minimal protease resistant tau unit of the Alzheimer's disease paired helical filament.EMBO J 12,365-70)。Von Bergen et al. determines minimum tau peptides/interaction motif (306-VQIVYK-311；SEQ ID NO:And Protein tau 146) On the second site (275-VQIINK-280) (SEQ ID NO:147), it forms β lamellas and is described as potential responsible initial Pathologic Protein tau aggregation PHF formation (von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow EM,Mandelkow E.2000.Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif((306)VQIVYK(311))forming beta structure.Proc Natl Acad Sci U S A 97:5129-5134)；EP 1214598；WO 2001/ 18546).For tau function collection of illustrative plates, referring to Fig. 2.Therefore, current strategies, which are intended to produce, does not destroy Protein tau in microtubule stabilization The resistant to aggregation medicine of intracellular effect in terms of change.

In addition, although under physiological conditions, Protein tau is considered as cell within a cell matter albumen, but intracellular Protein tau can Be released into ECS and promote neurodegeneration (G ó mez-Ramos, A., D í az-Hern á ndez, M., Cuadros, R.,Hernández,F.,and Avila,J.(2006).Extracellular tau is toxic to neuronal cells.FEBS Lett 580(20),4842-50).In fact, neurone loss is with neurofibrillary tangles (by tau eggs White composition) topography distribution in AD brains it is associated (West, M.J., Coleman, P.D., Flood, D.G., Troncoso,J.C.(1994).Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease.Lancet 344,769-72；Gómez-Isla,T., Price,J.L.,McKeel Jr,D.W.,Morris,J.C.,Growdon,J.H.,Hyman,B.T.(1996)。Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease.J Neurosci 16(14),4491-500；Gomez-Isla T,Hollister R,West H,Mui S, Growdon JH,Petersen RC,Parisi JE,Hyman BT.Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease.Ann Neurol 41:17-24 (1997)).In addition, total Protein tau and the horizontal of Phosphorylated tau increase in the celiolymph (CSF) of AD patient (Hampel,H.,Blennow,K.,Shaw,L.M.,Hoessler,Y.C.,Zetterberg,H.,Trojanowski,J.Q. (2010).Total and phosphorylated tau protein as biological markers of Alzheimer's disease.Exp Gerontol 45 (1), 30-40), and extracellular Protein tau has been described as in brain " ghost tangle (ghost tangle) " (Frost, B., Diamond, M.I. (2009) .The expanding realm of prion phenomena in neurodegenerative disease.Prion 3(2):74-7), show that intracellular tau is released It is put into ECS.In addition, extracellular Protein tau aggregation can enter cell and stimulate intracellular tau filaments (fibrillization) Protein tau for, producing pathologic Protein tau aggregation so as to further sow (seeding) to be used for Monomer (Frost etc., 2009).Insoluble Protein tau may act as that agent can be propagated and come with prion outside stressed cell for the research Sample loading mode spreads Protein tau lesion (Frost, B., Jacks, R.L., Diamond, M.I. (2009) throughout brain .Propagation of tau misfolding from the outside to the inside of a cell.J Biol Chem 284(19),12845-52；Frost et al.,2009；Frost,B.,Diamond,M.I.(2009).The expanding realm of prion phenomena in neurodegenerative disease.Prion 3(2): 74-7).The related extracellular and intracellular pathology of tau can be reduced by targetting abnormal tau.Referring to Eva Kontsekova, Norbert Zilka,Branislav Kovacech,Petr Novak,Michal Novak.2014.Targeting is for pathologic tau-tau phases Necessary to interaction structural determinant first for people tau vaccines (First-in-man tau vaccine) reduce Ah Tau oligomerizations and neurofibrillary degeneration in Er Cihai Mo's disease models.Alzheimer's Research&Therapy,6: 44.Therefore, to can by the formation of Protein tau outside block cell, promote its removing, or both reduce extracellular tau eggs Needs be present in white treatment and the treatment to reducing intracellular diseases Protein tau.To following in the conversion of pathologic Protein tau The understanding increase of molecular mechanism has turned on the possibility of selectively targeted pathologic Protein tau modification for therapeutic purposes.

Novak et al. international publication number WO 2013/041962 describes promote tau-tau to assemble in AD four The discovery in region, and the antibody for preventing tau from assembling by combining that four regions.

Although other researchs have described the antibody with reference to Protein tau sequence, and some in those antibody are it is reported that also can Disturb Protein tau aggregation and remove (Asuni AA, Boutajangout A, Quartermain D, Sigurdsson EM.Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements.J Neurosci 27:9115-9129 (2007)), but the anti-Protein tau antibody of monoclonal is there is no it is reported that just experience AD clinical examination Test.

Success of external source (mouse) monoclonal antibody in human treatment has been partially defined by people recipient for such a The obstruction of immunogenicity antiglobulin response caused by external therapeutic agent.These become the security of antibody and pharmaco-kinetic properties It is complicated.These challenges result in the development for the engineered antibody for carrying relatively low immune response risk.Constantly exploitation is each Kind patent engineering technology (such as chimerization, humanization, CDR transplanting, framework transplanting, affinity maturation, phage display, turn base Because of mouse) to promote the process.For nearest summary, referring to Safdari Y1, Farajnia S, Asgharzadeh M, Khalili M.Antibody humanization methods-a review and update.2013.Biotechnol Genet Eng Rev.29:175-86.doi:10.1080/02648725.2013.801235 with Almagro JC1, Fransson J.Humanization of antibodies.2008.Front Biosci.13:1619-33。

Major design retains the specificity and affinity of parental antibody, while the humanization with human constant region domain resists Body, it ideally less immunogenicity target will be presented to patient.Typical humanized antibody carries mouse or rat source Parental antibody complementary determining region (CDR) and mainly people source, but be often mutated to retain the combination of parental antibody Framework (FR) area of property and constant region.But antigen-binding affinity and specificity be not influence antibody bioactive and it is clinical into The single factor of work(.Activation of the improvement antibody to patients immune system is the key of the value of some humanized antibodies, and for Other antibody, it is target to reduce cell-mediated toxicity.Finally, the increased understanding to antibody structure and activity to study people Member is generally engineered the more advanced humanized antibody having the following properties that of more homogeneity by being mutated：More preferable antigen binding Characteristic (binding affinity, target-specific), effector function, stability, expression, purification properties, pharmacokinetics and medicine Effect is learned.These many improvement are important for the commercial viability for giving antibody.Sometimes, target binding affinity and special is being realized After property, it is necessary to which some amino acid being mutated in CDR or FR are to reduce neurological susceptibility of the humanized antibody to aggregation.Other when Wait, change (conversion or mutation) constant region to improve effector function.The these and other aspects of antibody function and activity continue Exploitation to the antibody of Clinical practice is challenged.Describe below and be engineered to have unexpected favorable property One group be directed to tau humanized antibody.Method and composition newly also provided below, its include these high degree of specificity and Efficient antibody, the antibody have specific recognition and binding of pathological tau, the ability for hindering it to assemble.It is all these anti- Body, method and composition are used to diagnosing and treating AD and associated tau protein lesion.

Summary of the invention

After the described in detail below and appended claims of disclosed embodiment have been read, of the invention these With other purposes, feature and advantage will be apparent.

Disclosed herein is the anti-tau antibody of humanization or its tau binding fragment, wherein the antibody or binding fragment include：

Respectively CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO.1,2,3 weight chain variable district are included, and is come from The framework (SEQ ID NO.71) of human immunoglobulin M 65092；

Respectively CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO.4,5,6 light chain variable district are included, and is come from Human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65)；With

Each from the heavy chain and constant region of light chain of human immunoglobulin(HIg)；And wherein described heavy chain framework selected from 9th, one or more of 21,27,28,30,38,48,67,68,70 and 95 position is substituted；The light chain framework is unsubstituted It is or substituted at 5；And wherein described position is according to Kabat.In some embodiments, antibody binding is selected from One or two of HQPGGG (SEQ ID NO.148), HVPGGG (SEQ ID NO.149) and HKPGGG (SEQ ID NO.150) Individual epitope.In some embodiments, all three epitopes of antibody binding.

This antibody and binding fragment also consider in the following form：Wherein described heavy chain 9 is occupied by P, 21 accounted for by P According to, 27 occupied by Y, 28 occupied by I, 30 occupied by T, 38 occupied by K, 48 occupied by I, 67 occupied by K, 68 Position is occupied by A, 70 occupied by L, and/or 95 are occupied by F.In one embodiment, light chain 5 is occupied by S.At some There was only 2 in embodiment, in this 11 positions to be occupied by so (as such).In some embodiments, this 11 positions In only 3 so occupied.In some embodiments, there was only 4 in this 11 positions so to be occupied.In some implementations There was only 5 in scheme, in this 11 positions so to be occupied.In some embodiments, there was only 6 in this 11 positions by this Sample occupies.In some embodiments, there was only 7 in this 11 positions so to be occupied.In some embodiments, this 11 There was only 8 in position to be occupied.In some embodiments, there was only 9 in this 11 positions so to be occupied.In some implementations There was only 10 in scheme, in this 11 positions so to be occupied.In some embodiments, all 11 in this 11 positions All so occupied.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO.148), HVPGGG (SEQ ID NO.149) and HKPGGG (SEQ ID NO.150) one or two epitope.In some embodiments, antibody binding is owned Three epitopes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is to be selected from RHA to RHM, Those of SEQ ID NO.13-25；And the sequence of light chain variable district is SEQ ID NO.26 (RKA).

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is to be selected from RHA to RHM, Those of SEQ ID NO.13-25；And the sequence of light chain variable district is SEQ ID NO.27 (RKB).

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.13, RHA, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.15, RHC, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17, RHE, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.18, RHF, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.19, RHG, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.20, RHH, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.21, RHI, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.22, RHJ, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.23, RHK, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.24, RHL, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.25, RHM, and the sequence of light chain variable district is SEQ ID NO.26, RKA.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.13, RHA, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.15, RHC, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17, RHE, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.18, RHF, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.19, RHG, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.20, RHH, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.21, RHI, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.22, RHJ, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.23, RHK, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.24, RHL, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.25, RHM, and the sequence of light chain variable district is SEQ ID NO.27, RKB.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.26, RKA, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.26, RKA, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.26, RKA, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.25, RHM, the sequence of light chain variable district is SEQ ID NO.26, RKA, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.26, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes.

The anti-tau antibody of humanization or its tau binding fragment are also contemplated, wherein the antibody or binding fragment include：

Comprising respectively SEQ ID NO.1,2,3 CDR-H1, CDR-H2 and CDR-H3 weight chain variable district, and come from The framework (SEQ ID NO.71) of human immunoglobulin M 65092；With

Comprising respectively SEQ ID NO.4,5,6 CDR-L1, CDR-L2 and CDR-L3 light chain variable district, and come from Human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65)；With

From human immunoglobulin(HIg), preferably IgG1 or IgG4 heavy chain and constant region of light chain.

It is further contemplated that anti-tau antibody or its tau binding fragment, wherein the antibody or binding fragment are comprising following Complete chain：

The heavy chain of amino acid sequence with any one of SEQ ID NO.28-40；With

The light-chain variable domain of amino acid sequence with any one of SEQ ID NO.26 and 27.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.

The heavy chain of amino acid sequence with any one of SEQ ID NO.43-55；With

The light-chain variable domain of amino acid sequence with any one of SEQ ID NO.27 and 58.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.

The heavy chain of amino acid sequence with SEQ ID NO.31；With

The light-chain variable domain of amino acid sequence with SEQ ID NO.57.In some embodiments, antibody binding is selected From HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or Two epitopes.In some embodiments, all three epitopes of antibody binding.

The heavy chain SEQ ID NO.32 for the amino acid sequence having；With

With any one of amino acid sequence light-chain variable domain SEQ ID NO.57.In some embodiments, resist Body combines and is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) One or two epitope.In some embodiments, all three epitopes of antibody binding.

The heavy chain of amino acid sequence with SEQ ID NO.31；With

The light-chain variable domain of amino acid sequence with SEQ ID NO.58.In some embodiments, antibody binding is selected From HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or Two epitopes.In some embodiments, all three epitopes of antibody binding.

The heavy chain of amino acid sequence with SEQ ID NO.32；With

The light-chain variable domain of amino acid sequence with SEQ ID NO.58 and 27.In some embodiments, antibody knot Conjunction is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one Individual or two epitopes.In some embodiments, all three epitopes of antibody binding.

The heavy chain of amino acid sequence with SEQ ID NO.46；With

The heavy chain of amino acid sequence with SEQ ID NO.47；With

The heavy chain of amino acid sequence with SEQ ID NO.46；With

The heavy chain of amino acid sequence with SEQ ID NO.47；With

Antibody is also contemplated, it is included：

Weight chain variable district, it includes SEQ ID NO 1,2,3 CDR-H1, CDR-H2 and CDR-H3, and is and SEQ ID NO.RHA,SEQ ID RHB,SEQ ID RHC,SEQ ID RHD,SEQ ID RHE,SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM, i.e. in SEQ ID NO.13-25 Any one at least 85% is identical；

With ripe light chain variable district, it includes SEQ ID NO 4,5,6 CDR-L1, CDR-L2 and CDR-L3 respectively, and And be identical with SEQ ID NO.26, RKA at least 85%, wherein the antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.In some embodiments, Antibody is chimeric.In some embodiments, antibody is humanization.

Antibody is also contemplated, it is included：

Weight chain variable district, includes SEQ ID NO 1,2,3 CDR-H1, CDR-H2 and CDR-H3, and is and SEQ ID NO:RHA,SEQ ID RHB,SEQ ID RHC,SEQ ID RHD,SEQ ID RHE,SEQ ID RHF,SEQ ID RHG,SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM, i.e. any in SEQ ID NO.13-25 Item at least 90% is identical；

With ripe light chain variable district, it includes SEQ ID NO 4,5,6 CDR-L1, CDR-L2 and CDR-L3 respectively, and And it is and SEQ ID NO:26, RKA at least 90% is identical, wherein the antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, all three epitopes of antibody binding.In some embodiments, antibody is chimeric.In some embodiments, antibody is Humanization.

Antibody is also contemplated, it is included：

Weight chain variable district, it includes SEQ ID NO 1,2,3 CDR-H1, CDR-H2 and CDR-H3, and is and SEQ ID NO:RHA,SEQ ID RHB,SEQ ID RHC,SEQ ID RHD,SEQ ID RHE,SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM, i.e. in SEQ ID NO.13-25 Any one at least 95% is identical；

With ripe light chain variable district, it includes SEQ ID NO 4,5,6 CDR-L1, CDR-L2 and CDR-L3 respectively, and And it is and SEQ ID NO:26, RKA at least 95% is identical, wherein the antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.In some embodiments, Antibody is chimeric.In some embodiments, antibody is humanization.

According to above three sections, such antibody and binding fragment also consider in the following form：Wherein heavy chain 9 is accounted for by P According to 21 are occupied by P, and 27 are occupied by Y, and 28 are occupied by I, and 30 are occupied by T, and 38 are occupied by K, and 48 are occupied by I, 67 Occupied by K, 68 are occupied by A, and 70 are occupied by L, and/or 95 are occupied by F.In one embodiment, light chain 5 is by S Occupy.In some embodiments, there was only 2 in this 11 positions so to be occupied.In some embodiments, this 11 positions There was only 3 in putting so to be occupied.In some embodiments, there was only 4 in this 11 positions so to be occupied.In some realities Apply in scheme, there was only 4 in this 11 positions is so occupied.In some embodiments, there was only 5 quilts in this 11 positions So occupy.In some embodiments, there was only 6 in this 11 positions so to be occupied.In some embodiments, this 11 There was only 7 in individual position so to be occupied.In some embodiments, there was only 8 in this 11 positions to be occupied.In some realities Apply in scheme, there was only 9 in this 11 positions is so occupied.In some embodiments, there was only 10 in this 11 positions So occupied.In some embodiments, all 11 in this 11 positions are so occupied.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO.148), HVPGGG (SEQ ID NO.149) and HKPGGG (SEQ ID NO.150 one or two epitope).In some embodiments, all three epitopes of antibody binding.

And antibody is also contemplated, it is included：

Weight chain variable district, it includes SEQ ID NO.1,2,3 CDR-H1, CDR-H2 and CDR-H3's, and is and SEQ ID NO:RHA,SEQ ID RHB,SEQ ID RHC,SEQ ID RHD,SEQ ID RHE,SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM, i.e. in SEQ ID NO.13-25 Any one at least 85% is identical；

With ripe light chain variable district, it includes SEQ ID NO 4,5,6 CDR-L1, CDR-L2 and CDR-L3 respectively, And it is and SEQ ID NO:27RKB at least 85% is identical, wherein the antibody binding is selected from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some implementations In scheme, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID 149) NO:150) one or two epitope.In some embodiments, all three epitopes of antibody binding.In some embodiments In, antibody is chimeric.In some embodiments, antibody is humanization.

And antibody is also contemplated, it is included：

Weight chain variable district, comprising SEQ ID NO.1,2,3 CDR-H1, CDR-H2 and CDR-H3, and it is and SEQ ID NO:RHA,SEQ ID RHB,SEQ ID RHC,SEQ ID RHD,SEQ ID RHE,SEQ ID RHF,SEQ ID RHG,SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM, i.e. any in SEQ ID NO.13-25 Item at least 90% is identical；

With ripe light chain variable district, it includes SEQ ID NO 4,5,6 CDR-L1, CDR-L2 and CDR-L3 respectively, and And it is and SEQ ID NO:27RKB at least 90% is identical, wherein the antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.In some embodiments, Antibody is chimeric.In some embodiments, antibody is humanization.

And antibody is also contemplated, it is included：

Weight chain variable district, it includes SEQ ID NO.1,2,3 CDR-H1, CDR-H2 and CDR-H3, and is and SEQ ID NO:RHA,SEQ ID RHB,SEQ ID RHC,SEQ ID RHD,SEQ ID RHE,SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM, i.e. in SEQ ID NO.13-25 Any one at least 95% is identical；

With ripe light chain variable district, it includes SEQ ID NO 4,5,6 CDR-L1, CDR-L2 and CDR-L3 respectively, and And it is and SEQ ID NO:27RKB at least 95% is identical, wherein the antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.In some embodiments, Antibody is chimeric.In some embodiments, antibody is humanization.

According to above three sections, such antibody and binding fragment also consider in the following form：Wherein heavy chain 9 is accounted for by P According to 21 are occupied by P, and 27 are occupied by Y, and 28 are occupied by I, and 30 are occupied by T, and 38 are occupied by K, and 48 are occupied by I, 67 Occupied by K, 68 are occupied by A, and 70 are occupied by L, and/or 95 are occupied by F.In one embodiment, light chain 5 is by S Occupy.In some embodiments, there was only 2 in this 11 positions so to be occupied.In some embodiments, this 11 positions There was only 3 in putting so to be occupied.In some embodiments, there was only 4 in this 11 positions so to be occupied.In some realities Apply in scheme, there was only 4 in this 11 positions is so occupied.In some embodiments, there was only 5 quilts in this 11 positions So occupy.In some embodiments, there was only 6 in this 11 positions so to be occupied.In some embodiments, this 11 There was only 7 in individual position so to be occupied.In some embodiments, there was only 8 in this 11 positions to be occupied.In some realities Apply in scheme, there was only 9 in this 11 positions is so occupied.In some embodiments, there was only 10 in this 11 positions So occupied.In some embodiments, all 11 in this 11 positions are so occupied.In some embodiments In, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149): 150) one or two epitope.In some embodiments, all three epitopes of antibody binding.

The disclosure also provides or considered the antibody in all paragraphs (summary of the invention) before this section, wherein the antibody or knot It is Fab, Fab', F (ab') to close fragment₂,Fd,scFv,(scFv)₂, or scFv-Fc.In some embodiments, this antibody knot Conjunction is selected from the one of HQPGGG (SEQ ID NO.148), HVPGGG (SEQ ID NO.149) and HKPGGG (SEQ ID NO.150) Individual or two epitopes.In some embodiments, all three epitopes of antibody binding.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody or binding fragment are IgG1, IgG2, IgG3, or IgG4 antibody.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, all three epitopes of antibody binding.

The disclosure also provides or considered the antibody in this section earlier paragraphs, resists wherein the antibody or binding fragment are IgG1 Body.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:149) and HKPGGG(SEQ ID NO:150) one or two epitope.In some embodiments, all three epitopes of antibody binding.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody or binding fragment are glycosylations 's.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:149) and HKPGGG(SEQ ID NO:150) one or two epitope.In some embodiments, all three epitopes of antibody binding.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody or binding fragment are with least 5x10^-7Affinity (K_D) combine Tau 151-391/4R.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.One In a little embodiments, all three epitopes of antibody binding.In some embodiments, antibody is chimeric.In some embodiment party In case, antibody is humanization.In some embodiments, binding affinity is measured by SPR.In some embodiments, Binding affinity is measured by ELISA..

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein with by American type culture collection The DC8E8 antibody of the hybridoma PTA-11994 secretions of (American Type Culture Collection) preservation is compared, institute State antibody or binding fragment with least 80% identical binding affinity, essentially identical binding affinity or preferably combine Affinity combination tau.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments, antibody binding institute There are three epitopes.In some embodiments, antibody is chimeric.In some embodiments, antibody is humanization.One In a little embodiments, binding affinity is measured by SPR.In some embodiments, binding affinity is measured by ELISA..

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody by U.S. typical case with being cultivated The DC8E8 antibody of the hybridoma PTA-11994 secretions of thing collection preservation competes the knot to tau at least one same epitope Close.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:149) and HKPGGG(SEQ ID NO:150) one or two epitope.In some embodiments, all three epitopes of antibody binding. In some embodiments, antibody is chimeric.In some embodiments, antibody is humanization.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody is caused by restructuring.One In a little embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG 149) (SEQ ID NO:150) one or two epitope.In some embodiments, all three epitopes of antibody binding.At some In embodiment, antibody is chimeric.In some embodiments, antibody is humanization.

In some embodiments, the antibody of preceding 4 paragraphs and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment are IgG1 isotypes.

In some of the other embodiments, the antibody and binding fragment of preceding 4 paragraphs in first 5 sections also cause weight chain variable The sequence in area is SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody It is IgG1 isotypes with binding fragment.

In some of the other embodiments, the antibody and binding fragment of preceding 4 paragraphs in first 6 sections also cause weight chain variable The sequence in area is SEQ ID NO.17, RHE, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody It is IgG1 isotypes with binding fragment.

In some of the other embodiments, the antibody and binding fragment of preceding 4 paragraphs in first 7 sections also cause weight chain variable The sequence in area is SEQ ID NO.25, RHM, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody It is IgG1 isotypes with binding fragment.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14RHB, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG4 same Kind type.

In some of the other embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16RHD, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG4 same Kind type.

In some of the other embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17RHE, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG4 same Kind type.

In some of the other embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.25RHM, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG4 same Kind type.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes.

In some embodiments, the antibody of preceding 4 paragraphs in first 8 sections and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.26, and RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 same Kind type.

In some embodiments, the antibody of preceding 4 paragraphs in first 9 sections and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.17, and RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 same Kind type.

In some embodiments, the antibody and binding fragment antibody and binding fragment of preceding 4 paragraphs in first 10 sections So that the sequence of weight chain variable district is SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and And constant region is IgG4 isotypes.

In some embodiments, the antibody and binding fragment antibody and binding fragment of preceding 4 paragraphs in first 11 sections So that the sequence of weight chain variable district is SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and And constant region is IgG4 isotypes.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody is in Chinese hamster ovary (CHO) recombinate and produce in cell line.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments In, all three epitopes of antibody binding.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody or binding fragment contain by Modify to change effector function, half-life period, proteolysis and/or glycosylated Fc areas.In some embodiments, antibody knot Conjunction is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one Individual or two epitopes.In some embodiments, all three epitopes of antibody binding.

It is 14 exemplary other embodiments in first 2 sections below：

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14RHB, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG1 same Kind type.

In some of the other embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16RHD, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG1 same Kind type.

In some of the other embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.17RHE, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG1 same Kind type.

In some of the other embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.25RHM, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG1 same Kind type.

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is that IgG4 isotypes obtain.

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody or binding fragment be modified with Regulation is selected from antibody dependent cellular cytotoxicity, complement-dependent cytotoxicity, serum half-life, bio distribution and and Fc The functional character that acceptor combines.In some embodiments, antibody binding is selected from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) one or two epitope.In some embodiments, antibody With reference to all three epitopes.

It is 14 exemplary other embodiments in earlier paragraphs below：

The disclosure also provides or considered the antibody in this section earlier paragraphs, wherein the antibody, which has, is equal to or more than 69 DEG C Thermostability temperature.

It is 14 exemplary other embodiments in earlier paragraphs below:

In another aspect of the present disclosure, it is contemplated that a kind of and another in antibody and/or binding fragment comprising the disclosure The composition of kind component.

In another aspect of the present disclosure, it is contemplated that comprising the antibody described in this section or binding fragment and pharmaceutically acceptable Carrier, diluent, the pharmaceutical composition of excipient or stabilizer.

It is 14 exemplary other embodiments in first 2 sections below：

The other form for the composition being additionally contemplates that before any, wherein the composition includes the antibody or bonding pad The freeze-dried powder of section.

The composition being additionally contemplates that before any, wherein the composition is formulated for into infusion or subcutaneous administration.

Any of these compositions are additionally contemplates that, it further (is also referred to as combined comprising second therapeutic agent (combination))。

It is 14 exemplary other embodiments in first 3 sections below：

In some in these compositions, therapeutic agent and antibody or binding fragment are chemically conjugated.

In some in these compositions, second therapeutic agent is used to prevent and/or treat AD.

Further contemplate second therapeutic agent and be selected from such as beta-amyloid peptide (beta-amyloid peptide) (such as N- ends Amyloid-beta-peptide), it may or may not be conjugated with other compounds, such as the diphtheria toxin of mutation；Other anti-tau resist Body, for the antibody of amyloid beta, such as bapineuzumab, solaneuzumab, gantenerumab, crenezumab With IVIG immunoglobulins, other immunotherapies of A beta oligomers are targetted, the compound of tau hyperphosphorylations is prevented, prevents tau The compound of oligomerization and aggregation or depolymerization tau oligomer (such as methyl thionin (methylthioninium), rember, or LMTX) and other targeting tau aggregations actively and passively immunotherapy；And its any pharmaceutically acceptable salt.

Further contemplate second therapeutic agent and be selected from amyloid-beta peptide aggregation inhibitor (for example, Tramiprosate), gamma-secretase Enzyme inhibitor (such as semagacestat) and gamma secretase modulators (tarenflurbil)；And its any it can pharmaceutically connect The salt received.

It is 14 exemplary other embodiments in first 4 sections below：

In some cases, second therapeutic agent is selected from acetylcholinesterase (acetylcholinesterase) inhibitor (for example, donepezil (donepezil), profit cuts down this bright (rivastigmine), galanthamine (galantamine), he gram Woods (tacrine), nutritious supplementary pharmaceutical), N-methyl-D-aspartate (NMDA) receptor antagonist (such as Memantine (memantine)), DNA repair inhibitors (for example, pirenzepine (pirenzepine) or its metabolin), transition metal chelate Agent, growth factor, hormone, nonsteroid anti-inflammatory drugs (NSAID), antioxidant, lipid lowering agent, selective di-phosphate ester enzyme level Agent, tau agglutination inhibitors, kinases inhibitor, resist mitochondria dysfunction Pharmacological inhibitors, neurotrophin, heat are stopped Gram protein inhibitor, lipoprotein associated phospholipase A₂Inhibitor, Memantine, anti-apoptotic compound, metal-chelator, DNA are repaired Inhibitor, 3-APS (3-amino-1-propanesulfonic acid) (3APS), 1,3- third disulfonic acid (1, 3-propanedisulfonate) (1,3PDS), secretion zymoexciter, beta-secretase inhibitor, inhibitors of gamma-secretase, β- Amyloid peptide, amyloid beta antibody, tau peptides, neurotransmitter, β-lamella disrupting agent (beta-sheet Breaker), anti-inflammatory molecular；And its any pharmaceutically acceptable salt.

It is 14 exemplary other embodiments in earlier paragraphs below:

In some embodiments, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

It is also contemplated that wherein second therapeutic agent is selected from the compound the (the particularly the 16th described in WO 2004/058258 With page 17), including medicine target (the 36-39 pages), alkanesulfonic acid and alkanol sulfuric acid (alkanolsulfuric Acid) (the 39-51 pages), anticholinesterase (the 51-56 pages), nmda receptor antagonist (the 56-58 pages), estrogen (the 58-59 pages), nonsteroid anti-inflammatory drugs (the 60-61 pages), antioxidant (the 61-62 pages) are peroxisome proliferator activated Acceptor (peroxisome proliferators-activated receptor) (PPAR) activator (the 63-67 pages), courage is solid Alcohol depressant (the 68-75 pages)；Amyloid inhibitors (the 75-77 pages), amyloid form inhibitor (77-78 Page), metal-chelator (the 78-79 pages), antipsychotic drug and antidepressants (the 80-82 pages), nutritious supplementary pharmaceutical (83-89 Page) and increase brain in bioactive substance availability compound (see the 89-93 pages) and prodrug (page 93 and 94)；And its appoint What pharmaceutically acceptable salt.

Composition is also contemplated, wherein second therapeutic agent is selected from the compound for preventing tau oligomerizations and aggregation and depolymerization tau is few The compound (such as methyl thionin, rember or LMTX) of aggressiveness.

It is 14 exemplary other embodiments in first 2 sections below

On the other hand, present disclose provides diagnostic reagent, it is included according to any one of claim 1-51 Antibody or binding fragment and carrier, diluent, excipient or stabilizer.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.RHB, and the sequence of light chain variable district is SEQ ID NO.RKA.Optionally, antibody and binding fragment are IgG1 Isotype.

In some other embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.16RHD, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and bonding pad Section is IgG1 isotypes.

In some other embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.17RHE, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and bonding pad Section is IgG1 isotypes.

In some other embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.25RHM, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and bonding pad Section is IgG1 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.26, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14RHB, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment are IgG4 isotypes.

In some other embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.16RHD, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and bonding pad Section is IgG4 isotypes.

In some other embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.17RHE, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and bonding pad Section is IgG4 isotypes.

In some other embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.25RHM, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and bonding pad Section is IgG4 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes.

In some embodiments of these diagnostic reagents, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes.

On the other hand, the disclosure provides the immunoconjugates with formula (A)-(L)-(C), wherein：(A) it is claim Any one of 1-51 antibody or its binding fragment；(L) it is joint；(C) is reagent (agent)；And wherein described joint (L) (A) to (C) is connected.In some cases, (C) is therapeutic agent, developer, can detect agent or diagnosticum.In some situations Under, (C) is therapeutic agent.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.14RHB, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment It is IgG1 isotypes.

In some other embodiments of these immunoconjugates, antibody and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.16RHD, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and combination Fragment is IgG1 isotypes.

In some other embodiments of these immunoconjugates, antibody and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.17RHE, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and combination Fragment is IgG1 isotypes.

In some other embodiments of these immunoconjugates, antibody and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.25RHM, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and combination Fragment is IgG1 isotypes.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.26, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes 's.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes 's.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.16, RHD, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes 's.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.17, RHE, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes 's.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.14RHB, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and binding fragment It is IgG4 isotypes.

In some other embodiments of these immunoconjugates, antibody and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.16RHD, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and combination Fragment is IgG4 isotypes.

In some other embodiments of these immunoconjugates, antibody and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.17RHE, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and combination Fragment is IgG4 isotypes.

In some other embodiments of these immunoconjugates, antibody and binding fragment also cause weight chain variable district Sequence is SEQ ID NO.25RHM, and the sequence of light chain variable district is SEQ ID NO.26RKA.Optionally, antibody and combination Fragment is IgG4 isotypes.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG1 isotypes 's.

In some embodiments of these immunoconjugates, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.14, RHB, the sequence of light chain variable district is SEQ ID NO.27, RKB, and constant region is IgG4 isotypes 's.

The present invention considers the nucleic acid molecules (RNA or DNA) for encoding above-disclosed any antibody and tau binding fragments. In one embodiment, such nucleic acid molecules include it is one or more selected from SEQ ID NO.96-124,141-142 and 127-140 nucleotide sequence.In other embodiments, such nucleic acid molecules include be selected from SEQ ID NO.96-124, One or more nucleotide sequences of any one at least 85% identical sequence in 141-142 and 127-140.In other embodiment party In case, such nucleic acid molecules include selected from any one in SEQ ID NO.96-124,141-142 and 127-140 at least One or more nucleotide sequences of 90% identical sequence.In other embodiments, such nucleic acid molecules are included and are selected from and SEQ One or more nucleic acid sequences of any one at least 95% identical sequence in ID NO.96-124,141-142 and 127-140 Row.In other embodiments, such nucleic acid molecules include selected from in SEQ ID NO.96-124,141-142 and 127-140 Any one at least 96% identical sequence one or more nucleotide sequences.In other embodiments, such nucleic acid molecules Comprising selected from one kind with least 97% identical sequence of any one in SEQ ID NO.96-124,141-142 and 127-140 Or multiple nucleic acids sequence.In other embodiments, such nucleic acid molecules, which include, is selected from and SEQ ID NO.96-124,141- One or more nucleotide sequences of any one at least 98% identical sequence in 142 and 127-140.In other embodiments In, such nucleic acid molecules include selected from any one in SEQ ID NO.96-124,141-142 and 127-140 at least 99% One or more nucleotide sequences of identical sequence.

In one embodiment, the disclosure considers nucleic acid molecules, and it includes the weight chain variable district for encoding anti-tau antibody (HCVR) or its tau binding fragment nucleotide sequence, wherein the HCVR or its fragment include：(i) it is derived from people's immune globulin White M65092 framework (SEQ ID NO.71), (ii) include the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, (iii) The HCVR CDR2 of amino acid sequence comprising SEQ ID NO.2, and (iv) include SEQ ID NO.3 amino acid sequence HCVR CDR3, and wherein described HCVR or its fragment include and SEQ ID NO:RHA to SEQ ID NO.RHM, i.e. SEQ Any of ID NO.13-25 amino acid sequence at least 98% identical amino acid sequence.In each of these embodiments In individual or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.In other embodiments In, antibody or its fragment include the heavy chain for further including IgG4 constant regions.

Nucleic acid molecules, it includes the light chain variable district (LCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65) is derived from, (ii) the LCVR CDR1 of the amino acid sequence comprising SEQ ID NO.4, (iii) include SEQ ID NO.5 amino acid sequence LCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its piece Section includes and SEQ ID NO:26, RKA, or SEQ ID NO.27, RKB amino acid sequence at least 98% identical amino acid sequence Row.In each of these embodiments or antibody or its fragment include the further heavy chain comprising κ constant regions Situation.

In one embodiment, the disclosure considers nucleic acid molecules, and it includes the weight chain variable district for encoding anti-tau antibody (HCVR) or its tau binding fragment nucleotide sequence, wherein the HCVR or its fragment include：(i) it is derived from people's immune globulin White M65092 framework (SEQ ID NO.71), (ii) include the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, (iii) The HCVR CDR2 of amino acid sequence comprising SEQ ID NO.2, and (iv) include SEQ ID NO.3 amino acid sequence HCVR CDR3, and wherein described HCVR or its fragment include and SEQ ID NO:RHA to SEQ ID NO.RHM, i.e. SEQ Any of ID NO.13-25 amino acid sequence identical amino acid sequence.In each of these embodiments, also may be used To be situation that antibody or its fragment include the further heavy chain comprising IgG1 constant regions.In other embodiments, antibody or Its fragment includes the heavy chain for further including IgG4 constant regions.

Nucleic acid molecules, it includes the light chain variable district (LCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65) is derived from, (ii) the LCVR CDR1 of the amino acid sequence comprising SEQ ID NO.4, (iii) include SEQ ID NO.5 amino acid sequence LCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its piece Section includes and SEQ ID NO:26, RKA, or SEQ ID NO.27, RKB amino acid sequence identical amino acid sequence.At this In each of a little embodiments or antibody or its fragment include the situation of the further heavy chain comprising κ constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:14, RHB amino acid sequence at least 98% identical amino acid sequence.At these In each of embodiment or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:16, RHD amino acid sequence at least 98% identical amino acid sequence.At these In each of embodiment or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:17, RHE amino acid sequence at least 98% identical amino acid sequence.At these In each of embodiment or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:25, RHM amino acid sequence at least 98% identical amino acid sequence.At these In each of embodiment or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:14, RHB amino acid sequence identical amino acid sequence.In these embodiments Each in or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:16, RHD amino acid sequence identical amino acid sequence.In these embodiments Each in or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:17, RHE amino acid sequence identical amino acid sequence.In these embodiments Each in or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its fragment include and SEQ ID NO:25, RHM amino acid sequence identical amino acid sequence.In these embodiments Each in or antibody or its fragment include the situation of the further heavy chain comprising IgG1 constant regions.

In some embodiments, any nucleic acid molecules that this section has just described cause antibody or its fragment to include further Include the heavy chain of IgG1 constant regions.

Nucleic acid molecules are further contemplated, it includes the light chain variable district (LCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its fragment include and SEQ ID NO:26, RKA amino acid sequence at least 98% identical amino acid sequence.At these In each of embodiment or antibody or its fragment include the situation of the further heavy chain comprising κ constant regions.

Nucleic acid molecules are further contemplated, it includes the light chain variable district (LCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its fragment include and SEQ ID NO:RKB amino acid sequence at least 98% identical amino acid sequence.In these realities Apply in each of scheme or situation that antibody or its fragment include the further heavy chain comprising κ constant regions.

Nucleic acid molecules are further contemplated, it includes the light chain variable district (LCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its fragment include SEQ ID NO:26, RKA amino acid sequence identical amino acid sequence.In these embodiments In each or antibody or its fragment include the situation of the further heavy chain comprising κ constant regions.

Nucleic acid molecules are further contemplated, it includes the light chain variable district (LCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its fragment include and SEQ ID NO:RKB amino acid sequence identical amino acid sequence.In these embodiments In each or antibody or its fragment include the situation of the further heavy chain comprising κ constant regions.

In some embodiments, any one of nucleic acid sequence encoding SEQ ID NO.13-25 (RHA to RHM) HCVR.

In some embodiments, nucleic acid sequence encoding SEQ ID NO.4, RHB HCVR.

In an embodiment of these nucleic acid, nucleic acid sequence encoding SEQ ID NO.16, RHD HCVR.

In an embodiment of these nucleic acid, nucleic acid sequence encoding SEQ ID NO.17, RHE HCVR.

In an embodiment of these nucleic acid, nucleic acid sequence encoding SEQ ID NO.25, RHM HCVR.

In an embodiment of these nucleic acid, nucleic acid sequence encoding SEQ ID NO.26, RKA, and appoint in 27, RKB The LCVR of one.

Nucleotide sequence is also contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino acid The HCVR CDR2 of sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR Or its fragment includes and SEQ ID NO:Any of RHA to SEQ ID NO.RHM (that is, 13-25) amino acid sequence is at least 98% identical amino acid sequence, and further such that the nucleotide sequence include under strict conditions with SEQ ID NO: The nucleotide sequence of any of RHA to SEQ ID NO.RHM (i.e. 96-108) complementary strand thereof, wherein the stingent hybridization Condition is included in 5xSSPE, 1%SDS, hybridizes in 1xDenhardts solution at 65 DEG C and is cleaned in 2xSSC, 1%SDS, and And 0.2xSSC is then used at 65 DEG C.

Nucleotide sequence is also contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino acid The HCVR CDR2 of sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR Or its fragment includes the amino acid sequence at least 98% identical amino acid sequence, and further with SEQ ID NO.14, RHB So that the nucleotide sequence includes the nucleotide sequence with SEQ ID NO.97, RHB complementary strand thereof under strict conditions, its Described in stringent hybridization condition be included in 5xSSPE, 1%SDS, in 65 DEG C of hybridization and in 2xSSC in 1xDenhardts solution, Cleaned in 1%SDS, and 0.2xSSC is then used at 65 DEG C.

Nucleotide sequence is also contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino acid The HCVR CDR2 of sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR Or its fragment includes the amino acid sequence at least 98% identical amino acid sequence, and further with SEQ ID NO.16, RHD So that the nucleotide sequence includes the nucleotide sequence with SEQ ID NO.99, RHD complementary strand thereof under strict conditions, its Described in stringent hybridization condition be included in 5xSSPE, 1%SDS, in 65 DEG C of hybridization and in 2xSSC in 1xDenhardts solution, Cleaned in 1%SDS, and 0.2xSSC is then used at 65 DEG C.

Nucleotide sequence is also contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino acid The HCVR CDR2 of sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR Or its fragment includes the amino acid sequence at least 98% identical amino acid sequence, and further with SEQ ID NO.17, RHE So that the nucleotide sequence includes the nucleotide sequence with SEQ ID NO.100, RHE complementary strand thereof under strict conditions, Wherein described stringent hybridization condition is included in 5xSSPE, 1%SDS, in 65 DEG C of hybridization and in 2xSSC in 1xDenhardts solution, Cleaned in 1%SDS, and 0.2xSSC is then used at 65 DEG C.

Nucleotide sequence is also contemplated, it includes the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino acid The HCVR CDR2 of sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR Or its fragment includes the amino acid sequence at least 98% identical amino acid sequence, and further with SEQ ID NO.25, RHM So that the nucleotide sequence includes the nucleotide sequence with SEQ ID NO.108, RHM complementary strand thereof under strict conditions, Wherein described stringent hybridization condition is included in 5xSSPE, 1%SDS, in 65 DEG C of hybridization and in 2xSSC in 1xDenhardts solution, Cleaned in 1%SDS, and 0.2xSSC is then used at 65 DEG C.

Nucleic acid molecules are further contemplated, it includes the light chain variable district (LCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its fragment include the amino acid sequence at least 98% identical amino acid sequence with SEQ ID NO.26, RKA；, and Further such that the nucleotide sequence includes the nucleotides with SEQ ID NO.109, RKA complementary strand thereof under strict conditions Sequence, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS, in 1xDenhardts solution 65 DEG C of hybridization and Cleaned in 2xSSC, 1%SDS, and 0.2xSSC is then used at 65 DEG C.

Nucleic acid molecules are further contemplated, it includes the light chain variable district (LCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its fragment include and SEQ ID NO:14, RKB amino acid sequence at least 98% identical amino acid sequence；, and Further such that the nucleotide sequence include under strict conditions with SEQ ID NO:The nucleotides of 110, RKB complementary strand thereof Sequence, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS, in 1xDenhardts solution 65 DEG C of hybridization and Cleaned in 2xSSC, 1%SDS, and 0.2xSSC is then used at 65 DEG C.

The nucleic acid molecules group comprising the first nucleic acid molecules and the second nucleic acid molecules is also contemplated, wherein：

First nucleic acid molecules are comprising the weight chain variable district (HCVR) or its tau binding fragment for encoding anti-tau antibody Nucleotide sequence nucleic acid molecules, wherein the HCVR or its fragment include：(i) it is derived from the frame of human immunoglobulin M 65092 Frame (SEQ ID NO.71), (ii) include the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID The HCVR CDR2 of NO.2 amino acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and Wherein described HCVR or its fragment include and SEQ ID NO:In RHA to SEQ ID NO.RHM (that is, SEQ ID NO.13-25) The amino acid sequence of any one at least 98% identical amino acid sequence；

And wherein described second nucleic acid molecules include the LCVR or its tau binding fragment that encode anti-tau antibody nucleosides Acid sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID are derived from NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 amino The LCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence.

Nucleic acid comprising the light chain variable district (LCVR) for encoding anti-tau antibody or the nucleotide sequence of its tau binding fragment Molecule, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65) is derived from, (ii) the LCVR CDR1 of the amino acid sequence comprising SEQ ID NO.4, (iii) include SEQ ID NO.5 amino acid sequence LCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its piece Section includes the amino acid sequence at least 98% identical amino acid sequence with SEQ ID NO.26, RKA；

And wherein described second nucleic acid molecules include the HCVR or its tau binding fragment that encode anti-tau antibody nucleosides Acid sequence, wherein the HCVR or its fragment include：(i) framework (the SEQ ID of human immunoglobulin M 65092 are derived from NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 amino The HCVR CDR2 of acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.3 amino acid sequence.

Nucleic acid comprising the light chain variable district (LCVR) for encoding anti-tau antibody or the nucleotide sequence of its tau binding fragment Molecule, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65) is derived from, (ii) the LCVR CDR1 of the amino acid sequence comprising SEQ ID NO.4, (iii) include SEQ ID NO.5 amino acid sequence LCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its piece Section includes and SEQ ID NO:27, RKB amino acid sequence at least 98% identical amino acid sequence；

And wherein described second nucleic acid molecules include the HCVR or its tau binding fragment that encode anti-tau antibody nucleosides The wherein described HCVR of acid sequence or its fragment include：(i) framework (SEQ ID NO.71) of human immunoglobulin M 65092 is derived from, (ii) the HCVR CDR1 of the amino acid sequence comprising SEQ ID NO.1, (iii) include SEQ ID NO.2 amino acid sequence HCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.3 amino acid sequence.

Any of encoding heavy chain variable region RHA-RHM (SEQ ID NO.13-25) nucleic acid molecules are also contemplated, its In those nucleotide sequences be respectively selected from SEQ ID NO.28-40, or any one being respectively selected from SEQ ID NO.43-55.Appoint What these nucleic acid molecules can combine with the nucleic acid molecules of next paragraph.

Nucleic acid molecules any in coding light chain RKA or RKB are also contemplated, wherein the nucleic acid molecules are respectively selected from SEQ ID NO.57 and 58.

Consider coding amino acid as disclosed herein or its tau binding fragment disclosed above, variable light, completely Light chain, variable heavy chain, all possible combination of the nucleic acid of entire heavy chain.

In some examples of these nucleic acid group, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described First nucleic acid molecules include coding SEQ ID NO.14, the RHB HCVR of anti-tau antibody or the nucleotides of its tau binding fragment Sequence, and second nucleic acid molecules include coding SEQ ID NO.26, RKA LCVR or the nucleotides of its tau binding fragment Sequence.

In some examples of these colonies, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described One nucleic acid molecules include coding SEQ ID NO.16, the RHD HCVR of anti-tau antibody or the nucleotides sequence of its tau binding fragment Row, and second nucleic acid molecules include coding SEQ ID NO.26, RKA LCVR or the nucleotides sequence of its tau binding fragment Row.

In some examples of these colonies, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described One nucleic acid molecules include coding SEQ ID NO.17, the RHE HCVR of anti-tau antibody or the nucleotides sequence of its tau binding fragment Row, and second nucleic acid molecules include coding SEQ ID NO.26, RKA LCVR or the nucleotides sequence of its tau binding fragment Row.

In some examples of these colonies, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described One nucleic acid molecules include coding SEQ ID NO.25, the RHM HCVR of anti-tau antibody or the nucleotides sequence of its tau binding fragment Row, and second nucleic acid molecules include coding SEQ ID NO.26, RKA LCVR or the nucleotides sequence of its tau binding fragment Row.

In some examples of these colonies, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described One nucleic acid molecules include coding SEQ ID NO.16, the RHD HCVR of anti-tau antibody or the nucleotides sequence of its tau binding fragment Row, and second nucleic acid molecules include coding SEQ ID NO.27, RKB LCVR or the nucleotides sequence of its tau binding fragment Row.

In some examples of these colonies, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described One nucleic acid molecules include coding SEQ ID NO.17, the RHE HCVR of anti-tau antibody or the nucleotides sequence of its tau binding fragment Row, and second nucleic acid molecules include coding SEQ ID NO.27, RKB LCVR or the nucleotides sequence of its tau binding fragment Row.

In some examples of these colonies, they include the first nucleic acid molecules and the second nucleic acid molecules, wherein described One nucleic acid molecules include coding SEQ ID NO.14, the RKB HCVR of anti-tau antibody or the nucleotides sequence of its tau binding fragment Row, and second nucleic acid molecules include coding SEQ ID NO.27, RKB LCVR or the nucleotides sequence of its tau binding fragment Row.

One group of carrier is also contemplated, each includes coding such as the heavy chain of antibody described in this section earlier paragraphs or combination The nucleic acid of fragment.Another group of carrier considered is comprising antibody light chain coding as described by the either segment in this section earlier paragraphs Or those of the nucleic acid of binding fragment.Another group of carrier considered is as the either segment in this section earlier paragraphs is retouched comprising coding The antibody light chain or binding fragment stated and such as that of the heavy chain of antibody described in this section earlier paragraphs or the nucleic acid of binding fragment A bit.

It has also contemplated that the host cell for including any of these carriers.In some embodiments, host cell is Protokaryon.In other embodiments, host cell is eucaryon.It has also contemplated that and include any nucleic acid molecules group described above Host cell.

In some embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.17, RHE, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.25, RHM, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.17, RHM, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

In some of the other embodiments of these host cells, antibody and binding fragment also cause the sequence of weight chain variable district Row are SEQ ID NO.17, RHM, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and combination Fragment is IgG1 or IgG4 isotypes.

Consider the nucleic acid of any considerations above, antibody, carrier and host cell and its respective composition be used as being directed to Under medicine：

Prevention or treatment AD or another tau lesions (tauopathy)；

Suffer from or be easy to treat in the subject with Alzheimer's or another tau lesions suffering from, suspecting Alzheimer's or another tau lesions；

Suffer from or be easy to slow down in the subject with Alzheimer's or another tau lesions suffering from, suspecting The progress of AD or another tau lesions；

Suffer from or be easy to improve in the subject with Alzheimer's or another tau lesions suffering from, suspecting AD or relative symptom；With

Suffer from or be easy to reduce in the subject with Alzheimer's or another tau lesions suffering from, suspecting The risk of AD or another tau lesions or delay AD or another tau lesion breaking-outs.

Another aspect of the present disclosure is the method for producing antibody or its tau binding fragment with reference to people tau, and it includes culture Any host cell just described so that express the nucleic acid and produce antibody or its tau binding fragment.

In some embodiments of this method, antibody and binding fragment also make it that the sequence of weight chain variable district is SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

In some other embodiments of this method, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some other embodiments of this method, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.17, RHE, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some other embodiments of this method, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.25, RHM, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some other embodiments of this method, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some other embodiments of this method, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.17, RHM, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some other embodiments of this method, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

On the other hand consider and suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect Subject in treat the method for Alzheimer's or another tau lesions, it includes applying treatment effectively to subject The composition for including any antibody or binding fragment described in this section of amount.

On the other hand consider promote tau aggregations from suffer from, suspects suffer from or be easy to suffer from Alzheimer's or The method removed in the brain of the subject of another tau lesions, it includes including this section using therapeutically effective amount to subject The composition of any antibody or binding fragment of description.

On the other hand consider to suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspects Slow down the method for the progress of AD or another tau lesions in subject, it includes applying including for therapeutically effective amount to subject The composition of any antibody or binding fragment described in this section.

On the other hand consider and suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect Subject in improve AD or relative symptom method, it include to subject apply therapeutically effective amount comprising in this section The composition of any antibody or binding fragment of description.

On the other hand consider treatment, prevention or reverse suffer from, suspect suffer from or be easy to suffer from Alzheimer's or The method of cognition in the subject of another tau lesions, it includes the including in this section using therapeutically effective amount to subject The composition of any antibody or binding fragment of description.

On the other hand consider and suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect Subject in reduce the risk of AD or another tau lesions or the method for delay AD or another tau lesions breaking-out, including to Subject applies the composition for including any antibody or binding fragment described in this section of therapeutically effective amount.

In some embodiments, these compositions, any of antibody and/or binding fragment are applied by injecting. In other embodiments, these compositions, any of antibody and/or binding fragment are applied by intravenous infusion.

It is also contemplated that embodiments below, wherein by these compositions, any of antibody and/or binding fragment with 0.1mg/kg body weight to the antibody or the dosage of binding fragment of 20mg/kg body weight and the frequency between weekly and monthly is applied to The subject, so as to treat the subject.In preferred embodiments, antibody or binding fragment are with 0.1mg/kg body weight Dosage to 10mg/kg body weight is applied.In some preferred embodiments, antibody or binding fragment are with these preceding dosages It is any at least three months, preferably at least six months, may at least 12 months, may be in the period of 24 months Using.In another embodiment, by these compositions, any of antibody and/or binding fragment are with 0.1mg/kg bodies Weight to 10mg/kg body weight antibody or binding fragment dosage and between weekly and monthly, frequency preferably once every two weeks The subject is applied to, so as to treat the subject.In another embodiment, by these compositions, antibody and/or Any of binding fragment is with the dosage of the antibody of 0.01mg/kg body weight to 100mg/kg body weight or binding fragment and weekly Frequency between monthly is applied to the subject, so as to treat the subject.

In some embodiments of both approaches, antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14, RHB, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment It is IgG1 or IgG4 isotypes.

In some other embodiments of both approaches, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and bonding pad Section is IgG1 or IgG4 isotypes.

In some other embodiments of both approaches, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.17, RHE, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and bonding pad Section is IgG1 or IgG4 isotypes.

In some other embodiments of both approaches, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.25, RHM, and the sequence of light chain variable district is SEQ ID NO.26, RKA.Optionally, antibody and bonding pad Section is IgG1 or IgG4 isotypes.

In some other embodiments of both approaches, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.16, RHD, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and bonding pad Section is IgG1 or IgG4 isotypes.

In some other embodiments of both approaches, antibody and binding fragment also cause the sequence of weight chain variable district It is SEQ ID NO.17, RHM, and the sequence of light chain variable district is SEQ ID NO.27, RKB.Optionally, antibody and bonding pad Section is IgG1 or IgG4 isotypes.

On the other hand, any treatment method that this section has just described also includes at least one type by being selected from the group Assessment monitor the therapeutic advance of subject：Mini mental status examination (Mini-Mental State Exam) (MMSE), Alzheimer's assesses scale-cognition (Alzheimer's Disease Assessment Scale-cognitive) (ADAS-COG) impression (Clinician Interview-Based Impression), based on clinician's talks (CIBI), neurology battery of tests (Neurological Test Battery) (NTB), dementia disability are assessed (Disability Assessment for Dementia) (DAD), clinical dementia grading-box summation (Clinical Dementia Rating-sum of boxes) (CDR-SOB), neuropsychopathy application form (Neuropsychiatric Inventory) (NPI), positron emission tomography (PET imagings) scanning (Positron Emission Tomography (PET Imaging) scan) and magnetic resonance imaging (MRI) scanning (Magnetic Resonance Imaging (MRI)scan).In one embodiment, the type of assessment is nerve test combination (NTB).In another embodiment, The type of assessment is mini mental status examination (Mini-Mental State Exam).

In some embodiments, the practice of these any methods is also included the other treatments of at least one of effective dose Agent is simultaneously or sequentially applied to the subject.

For example, in some of these embodiments, other therapeutic agents are selected from anti-apoptotic compound, metal-chelator, DNA repair inhibitors, 3-APS (3APS), 1,3- third disulfonic acid (1,3PDS), secrete zymoexciter, beta-secretase Enzyme inhibitor, inhibitors of gamma-secretase, beta-amyloid peptide, anti-amyloid beta antibody, neurotransmitter, β-lamella are broken Bad agent, anti-inflammatory molecular, and anticholinesterase；And its any pharmaceutically acceptable salt.

In other embodiments of these embodiments, anticholinesterase is Tacrine, and more how profit cuts down the bright of this, Piperazine is neat, galanthamine, or nutritious supplementary pharmaceutical (nutritive supplement)；And its any pharmaceutically acceptable salt.

In some other embodiment of these embodiments, other therapeutic agents are selected from beta-amyloid peptide (example Such as, N- ends amyloid-beta peptide), it may or may not be conjugated with other compounds, such as the diphtheria toxin of mutation；It is anti- The antibody of amyloid beta, such as bapineuzumab, solaneuzumab, gantenerumab, crenezumab, Ponezumab and IVIG immunoglobulins, other immunotherapies of A beta oligomers are targetted, prevent the chemical combination of tau hyperphosphorylations Thing, prevention tau oligomerizations and the compound (for example, methyl thionin, rember or LMTX) for assembling or promoting tau oligomer depolymerization With other actively and passively immunotherapies of targeting tau pathological forms (such as aggregation)；It is and its any pharmaceutically acceptable Salt.

Some in these other embodiments, other therapeutic agents be selected from amyloid-beta agglutination inhibitor (such as Tramiprosate), inhibitors of gamma-secretase (such as semagacestat) and gamma secretase modulators (tarenflurbil)；And its any pharmaceutically acceptable salt.

In some of these embodiments, other therapeutic agents be selected from acetylcholinesteraseinhibitors inhibitors (for example, it is more how piperazine Together, profit cuts down the bright of this, galanthamine, Tacrine, nutritious supplementary pharmaceutical), N-methyl-D-aspartate (NMDA) receptor antagonist (such as Memantine (memantine)), DNA repair inhibitors (for example, pirenzepine (pirenzepine) or its metabolin), Transition metal chelator, growth factor, hormone, nonsteroid anti-inflammatory drugs (NSAID), antioxidant, lipid lowering agent, selective phosphoric acid Diester enzyme inhibitor, tau agglutination inhibitors, kinases inhibitor, resist mitochondria dysfunction Pharmacological inhibitors, neurotrophy Albumen, heat shock protein inhibitors, platelet-activating factor acetylhydro-lase inhibitor, Memantine, anti-apoptotic compound, metal-chelating Agent, DNA repair inhibitors, 3-APS (3APS), 1,3- third disulfonic acid (1,3PDS), secretion zymoexciter, β-point Secrete enzyme inhibitor, inhibitors of gamma-secretase, beta-amyloid peptide, amyloid beta antibody, neurotransmitter, β-lamella breaks Bad agent, anti-inflammatory molecular；And its any pharmaceutically acceptable salt.

In some of these embodiments, other therapeutic agents are selected from BACE inhibitor；Muscarinic antagonist (muscarinic antagonists), anticholinesterase；Gamma-secretase inhibitors；Gamma secretase modulators；HMG-CoA Reductase inhibitor；Nonsteroid anti-inflammatory drugs；N-methyl-D-aspartate receptor antagonist；Anti-amyloid antibodies；Dimension life Plain E；Nicotinic acetyl choline receptor agonists (nicotinic acetylcholine receptor agonists)；CB1 by Body inverse agonists (CB1 receptor inverse agonist) or CB1 receptor antagonists；Antibiotic；Growth hormone promotees secretion Element；Histamine H 3 antagonists；AMPA activators；PDE4 inhibitor；GABA_AInverse agonists, amyloid aggregation inhibitor；Glycogen Synthase kinase beta inhibitor；The accelerator of α secretase activities；PDE-10 inhibitor, cholesterol absorption inhibitor and its any pharmacy Upper acceptable salt.

In some of these embodiments, other therapeutic agents are secondary antibodies.In other embodiments, second is anti- Body is selected from bapineuzumab, solaneuzumab, gantenerumab, crenezumab, ponezumab and IVIG and ball is immunized Albumen.

Also contemplate assess suffer from, suspects suffer from or be easy to suffer from Alzheimer's or another tau lesions by The method of examination person, methods described include any antibody or tau binding fragment described in detection this section and the life from subject The combination of the component of thing sample, wherein it is described combined with biological sample detection instruction subject in Alzheimer's or Another tau lesions.In some of these embodiments, biological sample is biopsy, CSF, blood, serum or blood plasma Sample.

Also contemplating any treatment method described in earlier paragraphs can be implemented with antibody or tau binding fragments, Wherein described antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.14, RHB, and light chain variable district Sequence be SEQ ID NO.26, RKA.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

Also contemplating any treatment method described in earlier paragraphs can be implemented with antibody or tau binding fragments, Wherein described antibody and binding fragment cause the sequence of weight chain variable district to be SEQ ID NO.16, RHD, and light chain variable district Sequence is SEQ ID NO.26, RKA.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

Also contemplating any treatment method described in earlier paragraphs can be implemented with antibody or tau binding fragments, Wherein described antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.17, RHE, and light chain variable district Sequence be SEQ ID NO.26, RKA.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

Also contemplating any treatment method described in earlier paragraphs can be implemented with antibody or tau binding fragments, Wherein described antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.25, RHM, and light chain variable district Sequence be SEQ ID NO.26, RKA.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

Also contemplating any treatment method described in earlier paragraphs can be implemented with antibody or tau binding fragments, Wherein described antibody and binding fragment also cause the sequence of weight chain variable district to be SEQ ID NO.16, RHD, and light chain variable district Sequence be SEQ ID NO.27, RKB.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

Also contemplating any treatment method described in earlier paragraphs can be implemented with antibody or tau binding fragments, Wherein described antibody and binding fragment cause the sequence of weight chain variable district to be SEQ ID NO.17, RHM, and light chain variable district Sequence is SEQ ID NO.27, RKB.Optionally, antibody and binding fragment are IgG1 or IgG4 isotypes.

Brief description

Fig. 1：The schematic diagram of six isotypes (isoform) of people tau.

Fig. 2：People tau (2N4R) schematic iunctional diagram；Individually disclose " VQIINK " and " VQIVYK " and be used as SEQ ID NO 147 and 146.

Fig. 3：Protein (the SEQ ID NO in DC8E8 kappa light chain variables area:And DNA sequence dna (SEQ ID NO 8):91).

Fig. 4：Protein (the SEQ ID NO of DC8E8 weight chain variable districts:And DNA sequence dna (SEQ ID NO 7):90).

Fig. 5：DC8E8 κ light chains germline analyzes (being respectively SEQ ID NO 167 and 168 with appearance order)

Fig. 6：DC8E8 heavy chain germlines analyze (being respectively SEQ ID NO 66 and 169 with appearance order)

Fig. 7：Chimeric and muroid DC8E8 and Tau 151-391/4R combination

Fig. 8：DC8E8 heavy chain Humanized strategies.Residue important in structure (dried meat ammonia is highlighted with black matrix font and italics Acid, cysteine and asparagine).The residue of black matrix line represents that retroversion is mouse residues.CDR region be lowercase and by Point in gauge outfit represents.Represent CDR's by the asterisk in gauge outfitInterior residue.Fig. 8 individually discloses SEQ to there is order ID NO 7,71 and 13-25.Notice that SEQ ID NO.71 (accession number is M65092 in IMGT databases) are shown as without before Lead peptide MDWTWRFLFVVAAVTGVQS (SEQ ID NO.174).

Fig. 9：The light chain humanized strategies of DC8E8 κ.Residue important in structure (dried meat ammonia is highlighted with black matrix font and italics Acid, cysteine and asparagine).The residue of black matrix line represents that retroversion is mouse residues.CDR region be lowercase and by Point in gauge outfit represents.Represent CDR's by the asterisk in gauge outfitInterior residue.Fig. 9 individually discloses SEQ to there is order ID NO.8,65 and 26-27..Notice that SEQ ID NO.65 (accession number is X72449 in IMGT databases) are shown as without before Lead peptide QLLGLLMLWVSGSSG (SEQ ID NO.175).

Figure 10：Humanization and chimeric DC8E8 and Tau 151-391/4R combination.With DC8E8 RKA or DC8E8 The antibody of DC8E8 RHA or the DC8E8 RHB of RKB coexpressions assembly coding is compared with the antibody binding for being completely fitted antibody.

Figure 11：Humanization and chimeric DC8E8 and Tau 151-391/4R combination：RKA versions.

Figure 12：Humanization and chimeric DC8E8 and Tau 151-391/4R combination：RKB versions.

Figure 13：Humanization and chimeric DC8E8 and Tau 151-391/4R combination：Vk versions are (light with what is be fitted together to Chain).

Figure 14：Humanization and chimeric DC8E8 and Tau 151-391/4R combination：RHM versions.

Figure 15：The heat endurance of candidate's humanization DC8E8 antibody：The antibody of purifying is heated 10 points at a temperature of instruction Clock, then it is cooled to 4 DEG C before Tau combinations ELISA is carried out.

Figure 16：The heat transfer analysis of humanization candidate's DC8E8 antibody of purifying：The candidate antibodies Tm of purifying measure and With chimeric and muroid DC8E8 comparison.

Figure 17：Pass through the binding kineticses of the DC8E8 antibody of surface plasmon resonance assay：KD is determined：Muroid DC8E8.

Figure 18：Pass through the binding kineticses of the DC8E8 antibody of surface plasmon resonance assay：K_DMeasure：Chimeric DC8E8

Figure 19：Pass through the binding kineticses of the DC8E8 antibody of surface plasmon resonance assay：K_DMeasure：Humanization DC8E8 RHD/RKA(AX004)

Figure 20：Pass through the binding kineticses of the DC8E8 antibody of surface plasmon resonance assay：K_DMeasure：Humanization DC8E8 RHE/RKA(AX005)

Figure 21：Pass through the binding kineticses of the DC8E8 antibody of surface plasmon resonance assay：K_DMeasure：Humanization DC8E8 RHD/RKB(AX016)

Figure 22：Pass through the binding kineticses of the DC8E8 antibody of surface plasmon resonance assay：K_DMeasure：Humanization DC8E8 RHE/RKB(AX017)；

Figure 23：Biacore result summaries

Figure 24：The antibody candidate aggregate analysis of full-length human：A.AX004(DA)；B.AX005(EA)；C.AX016 (DB)；D.AX017(EB).

Figure 25：Assess the solubility of the humanized antibody candidate of DC8E8 purifying：Dissolving spectrum in concentration process.

Figure 26：Assess the solubility of the humanized antibody candidate of DC8E8 purifying：Dissolving spectrum in concentration process：Concentration Binding activity afterwards.

Figure 27：Frost/thawing of humanization candidate antibodies stress be analyzed.

Figure 28：The thermal induction of the antibody candidate of full-length human stress be analyzed：A.AX004(DA)；B.AX005(EA)； C.AX0016(DB)；D.AX017(EB).

Figure 29：DC8E8 and (B) mouse DC8E8 is such as fitted together to by (A) of ELISA measure and mistake is unordered (misdisordered) the tau 151-391/4R and physiological tau 2N4R that truncate combination.(C) be fitted together to and mouse antibodies To the EC50 values for the Protein tau analyzed.

Figure 30：The tau of mouse and chimeric the DC8E8 truncation unordered with mistake is determined by surface plasma body resonant vibration 151-391/4R and total length physiological tau 2N4R balance combination binding constant.

Figure 31：(A) is determined by ELISA and is fitted together to DC8E8 and (B) mouse DC8E8 and the repetitive structure from albumen tau The combination of the tau peptides in domain.(C) be fitted together to and mouse antibodies are to the EC50 values for the tau peptides analyzed.

Figure 32：Chimeric DC8E8 suppresses pathologic tau-tau during tau filaments (fibrillization) determine in vitro Interaction.Conformation change and filament are undergone by the tau 151-391/4R of the unordered truncation of heparin-induced mistake, passed through Its degree of thioflavine T (Thioflavin) fluorescence measurement；Test is fitted together to DC8E8, and it prevents pathologic conformation change and tau-tau The ability of interaction.

Figure 33：Such as determined by ELISA, DC8E8 (IgG4 isotypes) humanization lead and chimeric DC8E8 and mistake Unordered tau 151-391/4R and total length physiological tau 2N4R combination.(A) humanized antibody AX004 combination；(B) people Source antibody A X005 combination；(C) humanized antibody AX016 combination；(D) humanized antibody AX017 combination.(E) it is fitted together to DC8E8 combination.(F) DC8E8 and humanization lead AX004, AX005, AX016, AX017 is fitted together to the tau eggs analyzed White EC50 values.

Figure 34：Such as determined by ELISA, DC8E8 (IgG1 isotypes) humanization lead and wrong unordered tau 151-391/4R and total length physiological tau 2N4R combination.(A) humanized antibody AX004 combination；(B) humanized antibody AX005 combination；(C) humanized antibody AX016 combination；(D) humanized antibody AX017 combination.(E) humanization lead AX004, AX005, AX016, AX017 are to the EC50 values for the Protein tau analyzed.

Figure 35：Surface plasma resonance (SPR) characterizes humanization DC8E8 lead AX004, AX005, AX016, AX017, With reference to mistake unordered tau151-391/4R and total length 2N4R.(A) IgG4 versions, (B) IgG1 versions.

Figure 36：Such as determined by ELISA, humanized antibody (IgG4 isotypes) is combined with the micro-pipe from albumen tau The combination of the tau peptides of duplicate block.(A) humanized antibody AX004 combination；(B) humanized antibody AX005 combination；(C) people source Change antibody A X016 combination；(D) humanized antibody AX017 combination.(E) it is fitted together to DC8E8 combination.(F) be fitted together to DC8E8 and Humanization version AX004, AX005, AX016, AX017 are to the EC50 values for the Protein tau analyzed.

Figure 37：Such as determined by ELISA, humanized antibody (IgG1 isotypes) is combined with the micro-pipe from albumen tau The combination of the tau peptides of duplicate block.(A) humanized antibody AX004 combination；(B) humanized antibody AX005 combination；(C) people source Change antibody A X016 combination；(D) humanized antibody AX017 combination.(E) be fitted together to DC8E8 and humanization version AX004, AX005, AX016, AX017 are to the EC50 values for the Protein tau analyzed.

Figure 38：Humanization DC8E8 leads suppress pathologic tau-tau phases in the tau filamentizations measure based on fluorescence Interaction.The unordered tau 151-391/4R experience conformation changes of induction mistake and filament, as surveyed by thioflavin T fluorometric It is fixed；Humanized antibody is added in being reacted to filamentization and tests its ability for preventing pathologic conformation change.The people of all tests Source antibody can suppress pathologic tau-tau aggregations.(A) pathology of the humanization DC8E8 leads induction of IgG4 isotypes Property aggregation suppression and (B) IgG1 isotypes humanized antibody induce pathological aggregation suppression.

Figure 39：Use humanization DC8E8 antibody (IgG1) immunohistochemical staining Alzheimer's brain.DC8E8 (A) and chimeric DC8E8 (B) identifies the neurofibrillar pathology of high load in people AD hippocampus (CA1).Humanized antibody AX004 (C) shown and DC8E8 identical staining patterns with AX016 (E).In general, humanized antibody AX005 (D) and AX017 (F) Identify pathological structures less in AD brains.Tool bar (tool bar)：100μm

Figure 40：Use humanization DC8E8 antibody (IgG4) immunohistochemical staining Alzheimer's brain.DC8E8 (A) and chimeric DC8E8 (B) identifies the neurofibrillar pathology of high load in people AD hippocampus (CA1).Humanized antibody AX004 (C) shown and DC8E8 identical staining patterns with AX016 (E).In general, humanized antibody AX005 (D) and AX017 (F) Identify pathological structures less in AD brains.Tool bar：100μm

Figure 41：Using humanization DC8E8 antibody (IgG1) immunohistochemical staining FTDP-17 brains (at R406W Tau is mutated).DC8E8 (A) and chimeric DC8E8 (B) identifies the neurofibrillar pathology of high load in people's entorhinal cortex.People source Change antibody A X004 (C) and AX016 (E) and show the staining pattern similar to DC8E8.In general, humanized antibody AX005 (D) Pathological structures less in FTDP-17 brains are identified with AX017 (F).Tool bar：100μm

Figure 42：Using humanization DC8E8 antibody (IgG4) immunohistochemical staining FTDP-17 brains (at R406W Tau is mutated).DC8E8 (A) and chimeric DC8E8 (B) identifies the neurofibrillar pathology of high load in people's entorhinal cortex.People source Change antibody A X004 (C) and AX016 (E) displayings and DC8E8 identical staining patterns.AX005 (D) and AX017 (F) nonrecognition Any pathological structures in FTDP-17 brains.Tool bar：100μm

Figure 43：Use humanization DC8E8 antibody (IgG1) immunohistochemical staining corticobasal degeneration.DC8E8(A) Substantial amounts of neuroglia tau pathology in people's caudate nucleus is identified with chimeric DC8E8 (B).Humanized antibody AX004 (C) and AX016 (E) displaying and DC8E8 identical staining patterns.In general, in humanized antibody AX005 (D) and AX017 (F) identification CBD brains Less pathological structures, but staining power is suitable with DC8E8.Tool bar：50μm

Figure 44：Use humanization DC8E8 antibody (IgG4) immunohistochemical staining corticobasal degeneration.DC8E8(A) Substantial amounts of neuroglia tau pathology in people's caudate nucleus is identified with chimeric DC8E8 (B).Humanized antibody AX004 (C) and AX016 (E) displaying and DC8E8 identical staining patterns.In general, in humanized antibody AX005 (D) and AX017 (F) identification CBD brains Less pathological structures, but staining power is suitable with DC8E8.Tool bar：50μm

Figure 45：Use humanization DC8E8 antibody (IgG1) immunohistochemical staining stein-leventhal syndrome.DC8E8 (A) and chimeric DC8E8 (B) identifies substantial amounts of neuroglia tau pathology in people's caudate nucleus.Humanized antibody AX004 (C) and AX016 (E) is shown and DC8E8 identical staining patterns.In general, humanized antibody AX005 (D) and AX017 (F) identifications are slightly Micro- less pathological structures.Staining power is suitable with DC8E8.Tool bar：50μm

Figure 46：Use humanization DC8E8 antibody (IgG4) immunohistochemical staining stein-leventhal syndrome.DC8E8 (A) and chimeric DC8E8 (B) identifies substantial amounts of neuroglia tau pathology in people's caudate nucleus.Humanized antibody AX004 (C) and AX016 (E) is shown and DC8E8 identical staining patterns.In general, humanized antibody AX005 (D) and AX017 (F) identifications are slightly Micro- less pathological structures.Staining power is suitable with DC8E8.Tool bar：50μm

Provide the amino acid sequence corresponding to people's tau isotypes in SEQ ID NO.151-156 respectively with appearance order：

Detailed description of the invention

Definition

Term " affinity " refer to molecule (such as antibody) single binding site gametophyte in connection (such as antigen) it Between noncovalent interaction total intensity.Equilibrium dissociation constant (K can typically be passed through_D) (or its back balance binding constant, K_A) Represent affinity of the molecule X to its gametophyte Y.Can by conventional method known in the art, including it is described herein those To measure affinity.See, for example, Pope ME, Soste MV, Eyford BA, Anderson NL, Pearson TW. (2009)J Immunol Methods.341(1-2):86-96 and method described herein.It is described below and is combined for measuring The certain illustrative and exemplary of affinity.

Term " amino acid " refers to amino acid naturally occur, modification and synthesis, and amino acid analogue and amino Acid mimic, it with the amino acid similar mode naturally occurred to work.The amino acid naturally occurred is by genetic code Those of coding, and those amino acid modified later, for example, hydroxy-proline, Gla, and O- phosphoric acid silks Propylhomoserin.Amino acid analogue refers to has identical basic chemical structure, i.e. α-carbon, carboxylic with reference to hydrogen with the amino acid naturally occurred Base, amino, and the compound of R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.This Class analog has the R group (for example, norleucine) of modification or the peptide backbone of modification, but the amino acid with naturally occurring is protected Hold identical basic chemical structure.Amino acid simulant refers to the structure with the general chemical constitution different from amino acid, still The compound to be worked in a manner of the amino acid to naturally occurring is similar.Suitable amino acid includes, but are not limited to send out in peptide 20 existing common amino acid naturally occurred and the sum of the natural generation prepared by organic synthesis or other metabolic pathways D- the and L- isomers for the amino acid that non-natural occurs.The example for the amino acid that such non-natural occurs includes, but are not limited to N- Acetylglucos amino-Serine, N- Acetylglucos amino-Serine, and O- phosphotyrosines.The amino acid bag of modification Include, but be not limited to hydroxy-proline, pyroglutamic acid, Gla, O- phosphoserines, azetidinecarboxylic acid, 2- amino oneself two Acid, 3- aminoadipic acids, Beta-alanine, alanine, 2-amino-butyric acid, 4-Aminobutanoicacid, 6-Aminocaproic Acid, 2- amino heptan Acid, 2- aminoisobutyric acids, 3- aminoisobutyric acids, 2- diaminopimelic acids, three-butyl glycine, 2,4- diaminoisobutyric acids, chain Element, 2,2'- diaminopimelic acids, 2,3- diaminopropionic acids, Ethylglycocoll, sarcosine, N- ethyl asparagines, High proline, oxylysine, allohydroxylysine, 3- hydroxy-prolines, 4- hydroxy-prolines, isodensmosine, not different bright ammonia Acid, N- methylalanines, sarcosine, N- methyl isoleucines, N- methyl amyls glycine, N- methylvalines, naphthalene Base alanine, norvaline, norleucine, ornithine, amyl group glycine, pipecolic acid and Thioproline.Term ammonia Base acid also includes the amino acid naturally occurred, and it is the metabolin in some biologies but is not used for incorporation into by genetic code encoding In protein.This amino acid includes, but are not limited to ornithine, D-Orn, and D-Arg.

" cytotoxicity of antibody dependent cellular mediation " and " ADCC " refer to cell-mediated reaction, wherein expression Fc acceptors (FcRs) nonspecific cytotoxic cells (such as natural killer (NK) cell, neutrophil leucocyte and macrophage) identification The antibody that is combined on target cell simultaneously then causes the cracking of target cell.For mediating ADCC primary cell NK cells only to express Fc γ RIII, but monocytes Fc γ RI, Fc γ RII and Fc γ RIII.In Ravetch and Kinet, Annu.Rev.Immunol 9:The FcR expression on hematopoietic cell is summarized in 457-92 (1991) table 3 of page 464.In order to The ADCC activity of purpose of appraisals molecule, external ADCC measure can be carried out, such as in U.S. Patent number 5,500,362 or United States Patent (USP) Measure described in numbers 5,821,337.For the measure useful effector cell include PMBC (PBMC) and Natural killer (NK) cell.Or or extraly, can in vivo, such as in animal model, such as Clynes PNAS (USA) 95:The ADCC activity of purpose of appraisals molecule in animal model disclosed in 652-656 (1998).

" back mutation " is the mutation introduced in the nucleotide sequence of encoding humanized antibody, and the mutation produces corresponding The amino acid of amino acid in parental antibody (for example, donor antibody, such as rodent antibody).Can be in the anti-of present disclosure Retain some Framework residues from parental antibody in body humanizing process substantially to retain the binding property of parental antibody, together When the possibility immunogenicity of gained antibody is minimized.In an embodiment disclosed herein, parental antibody is mouse Source.For example, people's Framework residues are changed over parent's muroid residue by back mutation.Can be with the reality of the Framework residues of back mutation Example includes, but are not limited to Typical residues, interface packaging residue, rare parent's residue close to binding site, " vernier area (Vernier Zone) " (its formed thereon be resident CDR platform) in residue (Foote＆Winter, 1992, J.Mol.Biol.224,487-499), and those residues close to CDR H3.

Term " chimeric " antibody refers to following antibody, and wherein the part of heavy chain and/or light chain from particular kind of with resisting Corresponding sequence in body is identical or homologous or belongs to specific antibodies species or subclass (for example, chimeric humanization, species conversion Antibody), while the remainder of chain is identical or homologous with the corresponding sequence in the antibody of another species or belongs to another Antibody type or subclass, and the fragment of this antibody-like, if they show desired bioactivity (U.S. Patent number 4,816, 567；With Morrison etc., Proc.Natl.Acad.Sci.USA, 81:6851-6855(1984)).In one embodiment, Term " chimeric antibody " refers to following monoclonal antibodies, and it includes the variable region from a kind of source or species, i.e. land, and comes At least a portion of the constant region of separate sources or species is come from, the antibody is typically prepared by recombinant DNA technology.Sometimes it is excellent Chimeric antibody of the choosing comprising muroid variable region and human constant region.Such muroid/people's chimeric antibody is that ball is immunized comprising coding muroid The production of the expression immunoglobulin gene of the DNA section of albumen variable region and the DNA section of encoding human immunoglobulin's constant region Thing.The other forms of " chimeric antibody " by the invention are following forms, wherein from the type or subclass of initial antibodies Modifications and changes its type or subclass.Such " chimeric " antibody is also referred to as " species switched antibodies ".For producing chimeric antibody Method include conventional recombinant DNA and Gene transfer techniques currently known in the art.See, e.g., Morrison, S.L., Deng Proc.Natl.Acad Sci.USA 81 (1984) 6851-6855；U.S. Patent number 5,202,238 and 5,204,244.

" competitive binding " is determined in following measure, immunoglobulin/antibody/combination under being tested in the measure Fragment suppresses reference antibody and common antigen, such as tau (for example, tau 151-391/4R) specific binding.Known many classes The competitive binding assay of type, such as：The direct or indirect radiommunoassay of solid phase (RIA), the direct or indirect enzyme of solid phase are immunized (EIA), interlayer competition assay are determined (referring to Stahli etc., Methods in Enzymology 9:242(1983))；Solid phase is straight Biotin-avidin EIA is met (referring to Stahli etc., Methods in Enzymology 9:242(1983)137: 3614(1986))；Sandwich assay that measure that solid phase directly marks, solid phase directly mark (referring to Harlow and Lane, Antibodies:A Laboratory Manual,Cold Spring Harbor Press(1988))；Use I¹²⁵Mark Solid phase directly marks RIA (referring to Morel etc., Mol.Immunol.25 (1):7(1988))；Direct biotin-the antibiont of solid phase Fibroin EIA (Cheung etc., Virology 176:546(1990))；The RIA directly marked.(Moldenhauer etc., Scand.J.Immunol.32:77(1990)).Generally, the measure is directed to use with the purifying antigen with reference to the surface of solids or carrying These any cell, the reference immunoglobulin of unlabelled test immunoglobulin and mark.Exempted from by determining in test In the presence of epidemic disease globulin Reverse transcriptase is measured with reference to the amount of the surface of solids or the mark of cell.Usually, the test is exempted from Epidemic disease globulin is present in excess.Usually, in the presence of antibody excess is competed, it will suppress the special of reference antibody and common antigen Property combine at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

" complement-dependent cytotoxicity " or " CDC " refers to the ability of the molecular cleavage target in the presence of complement.Pass through complement First component (C1q) of system combines originates complement activation pathway with the molecule (such as antibody) of isogeneic complexing.In order to Complement activation is assessed, CDC measure, such as Gazzano-Santoro etc., J.Immunol.Methods 202 can be carried out:163 (1996) described in.

As used herein, term " conjugated " refers to the functional group and second molecule from first molecule (for example, antibody) The key or chemical part that chemical reaction between the functional group of (for example, therapeutic agent or medicine) is formed.This generic key includes, but not Be limited to covalent bond and non-covalent bond, at the same such chemical part include, but are not limited to esters, carbonic ester, imines phosphate, hydrazone, Acetal, ortho esters, peptide bond, and oligonucleotides key." key of hydrolysis-stable " represents that key is substantially stable in water and is having Do not reacted under pH value with water, including but not limited to reaction extends the time in physiological conditions." hydrolytically unstable or can drop The key of solution " expression key is degradable in water or aqueous solution, including in such as blood." the unstable or degradable key of enzyme " Represent that key is degraded by one or more enzymes.Only by way of example, some PEG and related polymer include polymer backbone or PEG gathers Polymer backbone and provided herein is protein, polypeptide or peptide one or more terminal functional groups between linking group in Degradable key.Such degradable linkage includes, but is limited by PEG carboxylic acids or the PEG carboxylic acids and bioactivator of activation Alcohol radical reacts the ester bond to be formed, wherein such ester group typically hydrolyzes with release bioactive agent in physiological conditions.Other water Solve degradable key and include but is not limited to carbonic acid ester bond；Due to amine and the imine linkage of aldehyde reaction；It is anti-with phosphate group by alcohol The phosphoric acid ester bond that should be formed；Hydrazone key, it is the reaction product of hydrazides and aldehyde；Acetal key, it is the reaction product of acetaldehyde and alcohol； Original acid ester key, it is the reaction product of formic acid esters and alcohol；The peptide bond formed by amido, including but not limited to polymer is such as PEG End, and the carboxyl of peptide；With the oligonucleotides key formed by phosphoramidite group, include but is not limited to, the end of polymer, and The 5' hydroxyls of oligonucleotides.

Term " DC8E8 " refers to the antibody described in WO2013/041962, and by American Type Culture Collection The hybridoma of preservation under (American Type Culture Collection) Patent Deposit number PTA-11994 produces. Disclosed in WO2013/041962 is following discoveries, that is, specifically binds one of tau four previous unidentified functional areas Or multiple antibody (for example, DC8E8) can suppress the formation of pathological tau aggregation body, and tau a variety of pathology can be detected Property form, some is (for example, the pathologic monomer) formed earliest in disease, and the tau's does not identify that functional areas are selected from 268-HQPGGG-273(SEQ ID NO:148) (in first repetitive structure domain of Protein tau), 299-HVPGGG-304 (SEQ ID NO:149) (in second repetitive structure domain of Protein tau), 330-HKPGGG-335 (SEQ ID NO:150) (in the 3rd repetitive structure domain of Protein tau), and 362-HVPGGG-367 (SEQ ID NO:149) (the of Protein tau In four repetitive structure domains).It is wrong for people by immunohistochemistry (IHC) and enzyme linked immunosorbent assay (ELISA) (ELISA) screening Unordered tau II (Tau 151-391/4R) (are also referred to as tau Δs (1-150 in this application by mistake；392-441)/4R) caused by The generation of its monoclonal antibody special to people's conjugate spirals fibril (PHF) of hybridoma.Gained group includes the mouse of IgG1 subclass Monoclonal antibody (mAb) DC8E8.DC8E8 epitope mapping discloses it and combines the previous unidentified epitopes of upper four of people tau.This Outside, DC8E8 other functional analyses disclose each epitope and represent functional areas different in tau.(it is described as AD diagnosis in these regions With the new target drone for the treatment of) in tau functional areas, the functional areas of the tau are selected from 268-HQPGGG-273 (SEQ ID NO:148) (in first repetitive structure domain of Protein tau), 299-HVPGGG-304 (SEQ ID NO:149) (in tau eggs In second white repetitive structure domain), 330-HKPGGG-335 (SEQ ID NO:150) (tied in the 3rd repetition of Protein tau In structure domain), and 362-HVPGGG-367 (SEQ ID NO:149) (in the 4th repetitive structure domain of Protein tau).

Term " diagnosis (diagnosing) " as used herein and " diagnosis (diagnosis) " refer to following methods, technology Personnel can be assessed by methods described and whether even determine subject by given disease or situation, be in this case Ah Er Cihai Mo's diseases and related tau lesions.Technical staff is often diagnosed on the basis of one or more diagnostic indicators, Such as such as biomarker, its amount (including exist or lack) indicates presence, seriousness or the missing of situation.

Together with diagnosis, clinical disease monitoring and prognosis are also extremely to be concerned about and field interested.It is important to know The stage of AD progress and speed are to design maximally effective treatment.Can be that patient selects if more accurate prognosis can be carried out Suitable treatment, and less stringent treatment is selected in some cases.Measuring pathologic tau levels as disclosed herein can With to according to AD progress classification will benefit from particular treatment subject and can be more suitable with wherein optional or additional procedures Other subjects distinguish.

For example, DC8E8 can distinguish preclinical AD, clinical AD at initial stage and fully developed whole latter stage AD (referring to WO2013/ 041962).Pathologic tau early stage (tau monomers, disome)-Braak ' s stages I in the preclinical AD of DC8E8 displaying people Dyeing.The stage of antibody identification pathologic tau oligomer and the stage (entanglement) of pathologic tau polymer.Fully developed In Alzheimer's (whole latter stage-Braak ' s stage VI), DC8E8 mainly identifies neurofibrillary tangles, neuritic plaque and god The pathologic tau polymer of warp form.DC8E8 identifies all developing stage for formation of being tangled in Alzheimer's. DC8E8 identifications are tangled early stage of development-monomer, disome and oligomer stage early stage formed, and before late period oligomer, entanglement Stage, and advanced stages stage-intracellular of pathologic tau polymer and extracellular neuron tangle.

Therefore, " diagnosis " or " making diagnosis " as used herein further comprises making prognosis, and it can be based on pathology Property horizontal tau measurement provides prediction clinical effectiveness (carrying out or without therapeutic treatment), (or treatment is the suitable treatment of selection It is no effective), or the current treatment of monitoring and possible change treatment.

Antibody " effector function " refers to those bioactivity, and it can be attributed to Fc areas (native sequences Fc areas or the ammonia of antibody Base sequence variants Fc areas).The example of antibody mediated effect function combines including C1q；Complement-dependent cytotoxicity；Fc acceptor knots Close；The cytotoxicity (ADCC) of antibody dependent cellular mediation；Phagocytosis；With the downward of cell surface receptor (such as B cell by Body；BCR).

Term " epitope " or " antigenic determinant " refer to immunoglobulin or antibody (or its antigen-binding fragment) specificity knot Site on the antigen of conjunction.Epitope can be not attached to amino by adjacent amino acid or because the three-level of protein folds and what is abutted Acid is formed.Generally denaturing solvent is kept exposing by adjacent amino acids formed epitope, and the epitope formed is folded by three-level Generally lost when being handled with denaturing solvent.Epitope generally include often in unique spatial conformation at least 3,4,5,6,7,8, 9th, 10,11,12,13,14 or 15 amino acid.Determining the method for the space conformation of epitope includes such as x-ray crystallography and two Tie up nuclear magnetic resonance.See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, volume 66, G.E.Morris, edit (1996)." comformational epitope " is that antibody or its tau binding fragment are special with conformation The epitope that specific fashion combines.In the case of the epitope based on protein, with reference to the protein that can depend on carrying epitope Two level, three or four structure.In other words, the mode that antibody is in a manner of structure is special, tertiary structure is special, or level Four knot The special mode of structure combines.Comformational epitope is a kind of present in pathologic tau (for example, present in Tau 151-391/4R) Epitope.

There are antibody and tau binding fragment as described herein any one or more of following four tau sites to be used as it " epitope ", some or all of which are comformational epitopes：268-HQPGGG-273(SEQ ID NO:148) (the first of Protein tau In individual repetitive structure domain), 299-HVPGGG-304 (SEQ ID NO:149) (in second repetitive structure domain of Protein tau), 330-HKPGGG-335(SEQ ID NO:150) (in the 3rd repetitive structure domain of Protein tau), and 362-HVPGGG-367 (SEQ ID NO:149) (in the 4th repetitive structure domain of Protein tau).These epitopes herein be also referred to as QT1, QT2, QT3 and QT4.DC8E8 combines whole QT1-QT4；In fact, DC8E8 epitope is HXPGGG, wherein X is any amino Sour (SEQ ID NO.157).

Papain digestion of antibodies produces two identical antigen-binding fragments, is referred to as " Fab " fragment, each has single Individual antigen binding site, and remaining " Fc " fragment, its name reflect its ability easily crystallized (crystallizable fragment).Stomach Protease Treatment produces with two antigen binding sites and remains able to the F (ab') of crosslinking antigen₂Fragment." Fv " is Fab pieces The part for the heavy chain that section includes.It can also recombinate and produce these any fragments.The Fc parts of antibody and the effector function of antibody, Cytotoxicity (ADCC) and complement-dependent cytotoxicity or phagocytosis including antibody dependent cellular combine.In the Fc areas of antibody Change (for example, mutation or glycosylation change) can be used for adjusting its any effector function and increase its serum half-life and Other drugs dynamic metabolism property.

First constant domain (CH1) of Fab the fragments also constant domain containing light chain and heavy chain.Fab' fragments are because in heavy chain The c-terminus of CH1 domains with the addition of some residues (including one or more cysteines from antibody hinge region) and different In Fab fragments.Fab '-SH are herein defined as Fab ' name, and the wherein cysteine residues of constant domain carry at least one trip From sulfydryl.F(ab')₂Antibody fragment is initially produced as Fab' fragments pair, has hinge cysteine between them.Antibody piece Other chemical couplings of section are also known.

Term " Fc " as used herein includes polypeptide, and it includes the constant region of antibody, does not exempt from including first constant region Epidemic disease protein structure domain.Therefore Fc refers to IgA, IgD and IgG most latter two constant region immunoglobulin domains, and IgE and IgM Last three constant region immunoglobulin domains, and N- ends are to the flexible hinge of these domains.For IgA and IgM, Fc can include J chains.Immunoglobulin domains C γ 2 and C γ 3 (C γ 2 and C γ 3) and C γ are included for IgG, Fc Hinge between 1 (C γ 1) and C γ 2 (C γ 2).Although the border in Fc areas can change, human IgG heavy chain Fc areas typically limit Surely comprising residue C226 or P230 to its carboxyl terminal, wherein numbering according to EU numbering systems.For human IgG1, in an implementation Fc areas are limited comprising residue P232 to its carboxyl terminal herein in scheme, wherein numbering according to EU numbering systems (Edelman G M etc., (1969) Proc Natl Acad Sci USA, 63 (1):78-85).For example, generation or purge process in antibody In, or the nucleic acid for the heavy chain for passing through modified recombinant encoding antibody removes the C- terminal lysines in Fc areas (according to EU numbering systems 447 residues).Therefore, the component of complete antibody can include all K447 residues be removed antibody population, K447 residues not by The antibody population of removal, and there is the antibody population for the mixtures of antibodies for containing and not containing K447 residues.Fc can refer in separation The region or Fc polypeptide backgrounds, such as the region in antibody.Fc can be native sequences Fc or variant Fc.Replaced in Fc parts It is as known in the art (see, for example, Winter etc., U.S. Patent number that amino acid residue, which is changed, to change the effector function of antibody 5,648,260 and 5,624,821).One described in PCT application WO 93/10151 (being incorporated to from there through reference) is suitable Fc be the Fc areas that human IgG1's antibody is extended to from N- terminus hinge regions native C-tenninus single chain polypeptide.Another important Fc Polypeptide is U.S. Patent number 5,457,035 and Baum etc., and 1994, EMBO J.13:Fc mutains described in 3992-4001. The amino acid sequence of natural Fc sequence of the amino acid sequence of the mutain with being presented in WO 93/10151 is identical, except ammonia Base acid 19 changes into Ala from Leu, and amino acid 20 changes into Glu from Leu, and amino acid 22 changes from Gly For Ala.The affinity that mutain is reduced to Fc by body display.

Term " Fc acceptors " or " FcR " are used for the acceptor for describing the Fc areas of binding antibody.Preferable FcR is native sequences people FcR.Furthermore it is preferred that FcR be following one kind, its combine IgG antibody (γ acceptors) and including Fc. γ .RI, Fc. γ .RII and Fc. the acceptor of γ RIII subclass, including allelic variant and or these acceptors splicing form.Fc γ RII acceptors include Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" suppression acceptor "), it has similar ammonia mainly different in its cytoplasmic domains Base acid sequence.Activated receptor Fc γ RIIA contain the activation motif based on immunity receptor tyrosine in its cytoplasmic domains (ITAM).Suppress acceptor Fc γ RIIB and contain the suppression motif (ITIM) based on immunity receptor tyrosine in its cytoplasmic domains (referring to summary M.in Daeron, Annu.Rev.Immunol 15:203-234(1997)).In Ravetch and Kinet, Annu.Rev.Immunol 9:457-92(1991)；Capel etc., Immunomethods 4:25-34(1994)；With de Haas Deng J.Lab.Clin；Med.126:330-41 reviews FcR in (1995).Other FcR, it is included in following those that to be identified It is included in this paper term " FcR ".Term also includes neonatal receptor FcRn, and it is responsible for maternal IgG being transferred to fetus (Guyer etc., J.Immunol.117:587 (1976) and Kim etc., J.Immunol.24:249 (1994)), and adjust immune ball The stable state of albumen.

" Fv " and minimum antibody fragment, it contains complete antigen recognizing and antigen binding site.The region is by tight The dimer composition of one heavy chain of close Non-covalent binding and a light-chain variable domain.In the configuration, three of each variable domain Hypervariable region interacts to limit antigen binding site on the surface of VH-VL dimers.Generally, six hypervariable regions assign anti- Body antigen-binding specificity.However, even if single variable domain (or one of the Fv comprising only three hypervariable regions to antigen-specific Half) also there is the ability for identifying and combining antigen, although with the affinity lower than entire binding site.

As used herein, such as due to the somatic mutation naturally occurred or site directed mutation is deliberately introduced, comprising next From the weight of specific human germline immunoglobulin's sequence or humanized antibody and the specific Germline sequences of light chain variable " framework region " Weight or light chain variable framework region are compared, and can contain amino acid of differences.However, selected humanized antibody is in weight or light chain variable district The amino generally encoded on the amino acid sequence of framework region with the weight or light chain variable framework region of human germline immunoglobulin's gene Acid sequence has at least 90% homogeneity and contains following amino acid residues, when with other species (for example, muroid germline sequence Row) germ-line immunoglobulin amino acid sequence when comparing, humanized antibody is accredited as from people by it.In some situations In, humanized antibody preferably weight or light chain variable framework region amino acid sequence on germ-line immunoglobulin gene code The amino acid sequence of weight or light chain variable framework region has at least 90%, more preferably at least 95%, more preferably at least 96%, optimal The homogeneity of choosing at least 97%, especially at least 98%, most especially at least 99%.Generally, from the people of specific human germ line sequences The weight or light chain variable framework region of source antibody will be shown and the weight or light chain variable frame of human germline immunoglobulin's gene code The amino acid sequence in frame area is no more than 11 amino acid, preferably more than 5, or even more preferably no more than 4,3,2 or 1 not Together.

" human effector cell " is the one or more FcR of expression and performs the granulocyte of effector function.Preferably, carefully Cellular expression at least Fc γ RIII and perform ADCC effector functions.Mediating the example of ADCC people's granulocyte includes peripheral blood Monocyte (PBMC), natural killer (NK) cell, monocyte, cytotoxic T cell and neutrophil leucocyte；It is preferred that PBMC and NK cells.Can be from its natural origin, such as the separation effect cell from blood as described herein or PBMC.

" human germ line sequences " are naturally found in ethnic group.The combination of those germ line genes produces antibody diversity.Antibody The germline antibody sequence of light chain comes from stick-in-the-mud's germline κ or λ v- genes and j- genes.Equally, sequence of heavy chain from germline v-, D- and j- genes (LeFranc, M-P, and LeFranc, G, " The Immunoglobulin Facts Book " Academic Press,2001).In the presence of the publicly available well-known database for all known Germline sequences.

The terms " hinge " or " hinge area " or " antibody hinge region " include following flexible polypeptides, its include antibody or Amino acid between first of its tau binding fragment and second constant domain.Present document relates to " hinge area " be that length is 6- The sequence area of 62 amino acid, is existed only in IgA, IgD and IgG, and it includes the cysteine residues for bridging two heavy chains. In one embodiment, in structure, IgG CH1 domains end at EU positions 220, and IgG CH2 domains start from Residue EU positions 237.Therefore, for IgG, in one embodiment, antibody hinge is defined herein as including position 221 (D221 in IgG1) to 231 (A231 in IgG1), wherein numbering according to EU numbering systems.

Term " humanized antibody " refers to following antibody, wherein having modified framework region (FR) and/or " complementary determining region " (CDR) with the CDR of the immunoglobulin comprising not homospecificity (mouse) compared with the CDR of parental immunoglobulin (people). In embodiment, muroid CDR is transplanted in the framework region of human antibody, to prepare " humanized antibody ".In another embodiment In, by people's framework " transplanting " or mouse antibodies are cut into, retains the CDR of mouse antibodies and replaces its frame using the framework of people source Frame.Transplanting and shearing can be completed by a variety of recombinant DNA technologies, including PCR and mutagenesis.A variety of people sources in this area be present Change method (for example, CDR transplanting, remodeling, transgenic animals, combinatorial libraries).See, for example, Riechmann, L., etc. Nature 332(1988)323-327；Neuberger, M.S., etc. Nature 314 (1985) 268-270；Sastry L,Alting- Mess M,Huse WD,Short JM,Sorge JA,Hay BN,Janda KD,Benkovic SJ,Lerner RA(1989) Cloning of the immunological repertoire in for generation of monoclonal catalytic antibodies:construction of a heavy chain variable region-specific cDNA library.Proc Natl Acad Sci USA 86,5728-5732；With Huse WD, Sastry S, Iverson SA,Kang AS,Alting-Mees M,Burton DR,Benkovic SJ,Lerner RA(1989)Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.Science 246,1275-1281.Humanized antibody due to another antibody of genetic modification with cause its more like People, while retain its original antigen binding property.Presta,L.G.Engineering of therapeutic antibodies to minimize immunogenicity and optimize function.Advanced Drug Delivery Reviews, volume 58, Issues 5-6:640–656(2006).Select people's variable domain (light chain and heavy chain) stand-by It is extremely important to reducing antigenicity in preparing humanized antibody.It is variable for known people according to so-called " best fit " method The library of domain sequence or the sequence of the library screening rodent antibodies variable domain of human germ line sequences.Then can by with rodent The most similar human sequence of sequence receives as people's framework region (Sims etc., J.Immunol.1993 for humanized antibody；151: 2296 et seq.；Chothia etc., Chothia and Lesk, J.Mol.Biol.1987；196:901-917).Another method makes With specific framework region, it is from light chain or the consensus sequence of all human antibodies of the specific subgroup of heavy chain.Identical framework can For some different humanized antibodies (Carter etc., PNAS USA, 1992；89:4285 et seq.；Presta etc., J Immunol 1993；151:2623 et seq.).Designed for reducing its other party of the immunogenicity of antibody molecule in people patient Method includes the antibody of modification (see, for example, U.S. Patent number 6,797,492 and the He of U.S. Patent Application Publication 20020034765 20040253645) antibody of modification and by t cell epitope is analyzed and removes (see, for example, U.S. Patent Application Publication 20030153043 and U.S. Patent number 5,712,120).

The particularly preferred CDR of humanized antibody as described herein corresponds to mouse monoclonal DC8E8 antibody, i.e. SEQ ID NO.1-6 CDR sequence.The humanized antibody and its tau as described herein prepared by recombination method or any other method is tied Close fragment copy (such as immunoglobulin with identical heavy chain or light chain variable district, as those described herein) (except Naturally occurring antibody) and the term in the range of humanized antibody or its tau binding fragment.

When used herein, term " hypervariable region " refers to the amino acid residue of the antibody of responsible antigen binding.In an implementation In scheme, according to Kabat, hypervariable region generally comprises the amino acid residue from " complementary determining region " or " CDR ", and (such as light chain can Residue 24-34 (L1), 50-56 (L2) in the variable domain and 31-35 (H1) in 89-97 (L3) and heavy chain variable domain, 50-65 (H2) With 95-102 (H3)；Kabat etc., Sequences of Proteins of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or From " hypervariable loop " those residues (such as residue 26-32 (L1), 50-52 (L2) and 91-96 (L3) in light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in heavy chain variable domain；Chothia and Lesk J.Mol.Biol.196: 901-917(1987))。

" framework region " or " FR " residue is those variable domain residues in addition to some hypervariable region residues being defined herein. In numbering system unique IMGT, conservative amino acid has an identical position all the time, such as Cys2 3 (1st-CYS), Tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP).See, for example, Lefranc M.-P., Immunology Today 18,509 (1997)；Lefranc M.-P.,The Immunologist,7,132-136(1999)；Lefranc,M.-P.,Pommié,C.,Ruiz,M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev.Comp.Immunol.,27,55-77(2003).In another embodiment, numbering unique IMGT provides framework region (FR1-IMGT:Position 1-26, FR2-IMGT:39-55、FR3-IMGT:66-104 and FR4-IMGT:118-128) determined with complementation Determine area：CDR1-IMGT:27-38、CDR2-IMGT:56-65 and CDR3-IMGT:105-117 standardization limits.Blank represents Unappropriated position, CDR-IMGT length (being shown between bracket and by a separation, such as [8.8.13]) become important Information.Numbering unique IMGT is used in 2D graphic representations, is named as IMGT Colliers de Perles.See, for example, Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53,857-883 (2002)；Kaas, Q. and Lefranc, M.- P.,Current Bioinformatics,2,21-30(2007).It is also used for representing 3D structures.See, for example, IMGT/ 3Dstructure-DB Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl.Acids.Res.,32,D208-D210(2004).Framework or FR residues are the high changes in bracket Those variable domain residues outside area.

Using Kabat numbering systems, actual linear amino acid sequence can contain corresponding to shortening or be inserted into variable Less or additional amino acid in the FR or HVR in domain.For example, heavy chain variable domain can be included in it is single after H2 residue 52 The residue of amino acid insertion insertion (according to Kabat residue 52a) and after heavy chain FR residue 82 is (such as according to the residual of Kabat Base 82a, 82b and 82c etc.).It can be surveyed by being compared on the homology region of antibody sequence with " standard " Kabat numbered sequences Surely the Kabat numberings of the residue of given antibody.

Term " immunogenicity " as used herein or the finger of immunogene respond to the antibody for applying medicine.Using fixed Amount and qualitative determination obtain be directed to provided herein is antibody and tau binding fragments immunogenicity be used for detect be directed to biofluid Described in treatment albumen, polypeptide and peptide antibody.Such measure includes, but are not limited to radiommunoassay (RIA), enzyme linked immunological Determining adsorption (ELISA), luminescent immunoassay (LIA), and fluorescence immunoassay (FIA).The analysis of the immunogenicity include than Compared with apply provided herein is antibody and tau binding fragments after antibody response with using randomized controlled treatment albumen, polypeptide or peptide or passing Send medium or deliver the antibody response after buffer solution.

" light chain " of antibody from any invertebrate species can be divided according to the amino acid sequence of their constant domains Send as one kind in two significantly different types, referred to as κ (κ) and λ (λ).

" natural antibody " is usually about 150,000 be made up of light (L) chain of two identicals and two identical weight (H) chains The heterotetrameric glycoproteins of dalton.Every light chain is combined with heavy chain by a covalent disulfide bonds, and the quantity of disulfide bond exists It is different between the heavy chain of different Immunoglobulin Isotypes.Every heavy chain and light chain also have two sulphur in the chain of aturegularaintervals Key.Each heavy chain has a variable domain (VH) at one end, is then multiple constant domain.Each light chain has one at one end Individual variable domain (VL), and there is constant domain in its other end.The constant domain of light chain aligns with the first of heavy chain constant domain, and Light-chain variable domain aligns with the variable domain of heavy chain.Believe that specific amino acid residue forms boundary between light chain and heavy chain variable domain Face.

Term " nucleic acid " as used herein is intended to include DNA molecular and RNA molecule.Nucleic acid molecules can be single-stranded or double Chain, but preferably double-stranded DNA.

Two sequences to be compared after two " percentage identity " expression optimal comparisons between nucleic acid or amino acid sequence The percentage of identical nucleotides or amino acid residue between row, the percentage be it is pure statistically and between two sequences Difference random distribution along its length.Two nucleic acid or amino acid sequence are carried out conventionally by comparative sequences after optimal comparison Comparison, the comparison can by section or by using " comparing window " and carry out.In addition to craft relatively, for what is compared Sequence it is optimal than Smith and Waterman (1981) [Ad.App.Math.2 can be passed through by following progress:482] office Portion's homology algorithm, pass through Neddleman and Wunsch (1970) [J.Mol.Biol.48:Local homology algorithm 443], Pass through Pearson and Lipman (1988) [Proc.Natl.Acad.Sci.USA 85:2444] search for similarity method or logical Cross computer software (Wisconsin Genetics the Software Package, Genetics using these algorithms Computer Group, 575 Science Dr., Madison, WI, in GAP, BESTFIT, FASTA and TFASTA or pass through Comparison software BLAST NR or BLAST P).For example, it is at least about 75-100 continuous that sequence identity, which may reside in length, In the region of unit, length is in the region of at least about 50 sequential cells, or is not known when illustrating, across the complete of peptide sequence Whole sequence.

By comparing two percentage identities between nucleic acid or amino acid sequence of sequencing of optimal comparison, wherein Nucleic acid to be compared or amino acid sequence can have compared with for the reference sequences of optimal comparison between two sequences to be added Add or lack.Between determining two sequences, such as between two complete sequences on identical amino acid nucleotides or residue Number of positions calculate percentage identity, same position quantity divided by compare window in position sum and result is multiplied by 100 To obtain the percentage identity between two sequences.In one embodiment, using passing through Kabat numbering convention high specifics To antibody sequence measure antibody between Percent sequence identity.After comparison, if subject's antibody district is (for example, heavy chain Or the complete ripe variable region of light chain) compared with the same area of reference antibody, then subject and reference antibody area Between Percent sequence identity be number of positions in subject and reference antibody area shared by same amino acid divided by two The sum of the comparison position in region is multiplied by 100 to be converted into percentage, and its Vacancy disregards number.

For example, can be in network address http:The upper available BLAST journeys of //www.ncbi.nlm.nih.gov/gorf/bl2.html Sequence, " sequences of BLAST 2 " (Tatusova etc., " the sequences-a new tool for comparing of Blast 2 protein and nucleotide sequences”,FEMS Microbiol.,1999,Lett.174:247-250) can be with (particularly parameter " open gap penalty " is used with default parameters:5, and " extension gap penalty ":2；Selected matrix is, for example, journey Sequence suggestion " matrixes of BLOSUM 62 ")；The percentage identity between two sequences is directly calculated by described program to carry out Compare.

For showing at least 80%, such as the amino of 85%, 90%, 95% and 98% homogeneity with reference amino acid sequence Acid sequence, preferable example are included containing reference sequences, some modifications, the missing of in particular at least one amino acid, addition or Substitution, those for truncating or extending.It is preferably following to take in the case of the one or more conservative or nonconserved amino acids of substitution Generation, wherein the amino acid substituted is by " equivalent " amino acid substitution.Herein, statement " equivalent amino acid " is meant that to take For structural amino acid once not changing any amino of the bioactivity of corresponding antibodies and those instantiations defined below Acid.

Can biology on the structural homology of its amino acid substituted with them or between issuable multiple antibody Equivalent amino acid is determined in the comparison test result of activity.As non-limiting examples, table 1 below outline may carry out but not Cause the possibility substitution that the bioactivity of corresponding modified antibodies significantly changes；The opposite the same terms that are substituted in are possible naturally 's.

Original Residue	Substitution
		Ala(A)	Val、Gly、Pro
Arg(R)	Lys、His
		Asn(N)	Gln
Asp(D)	Glu
		Cys(C)	Ser
Gln(Q)	Asn
		Glu(E)	Asp
Gly(G)	Ala
		His(H)	Arg
Ile(I)	Leu
		Leu(L)	Ile、Val、Met
Lys(K)	Arg
		Met(M)	Leu
Phe(F)	Tyr
		Pro(P)	Ala
Ser(S)	Thr、Cys
		Thr(T)	Ser
Trp(W)	Tyr
		Tyr(Y)	Phe、Trp
Val(V)	Leu、Ala

Table 1

In the context of the disclosure, term " pathologic tau " and " disease tau " includes pathologic tau conformers With structure and including all following：Tau types IA, IB, IIA and IIB (are described in detail in WO2004/007547 A2), disorderly , the soluble tau that the tau that mistake is unordered (monomer, disome, three bodies, oligomer), mistake are unordered, flesh aminoacyl insolubility Tau, extracellular tau deposits, tau aggregations, conjugate spirals fibril, neurofibrillar pathology, including neurofibril are damaged, twined Knot, line, fibrillation, aixs cylinder orbicule, truncate tau and total length tau hyperphosphorylation form, or with AD or another tau disease (it can be examined the related tau of change (tauopathy) any other form by antibody as described herein or tau binding fragments Survey).Tau 151-391/4R (also referred to as tau Δs (1-150；92-441)/4R) represent pathologic tau form.

Term " pharmaceutically acceptable " means biologically or to be pharmacologically adapted to be used in animal or people in vivo, and excellent Choosing mean by federal or state government management organization ratify or be listed in American Pharmacopeia or other generally approve be used for animal or In the more specific pharmacopeia in people.

As used herein, term " recombinant host cell " (or simply " host cell "), which is intended to refer to, wherein has been introduced into restructuring The cell of expression vector.It should be understood that such term is intended to refer not only to the offspring that specific subject cell also refers to such cell.Cause To be influenceed due to mutation or environment, some modifications can occur in subculture, and such offspring actually may be with mother cell not phase Together, but it is included in the range of term as used herein " host cell ".

Term " another tau lesions " is included in all god occurred in patient's brain with the anomaly pattern of microtubule bindin Through disease.Term includes but is not limited to following disease：Alzheimer's,Disease Disease, Britain dull-witted (British dementia), Denmark dull-witted (Danish dementia), Pick's disease (Pick ' s Disease), stein-leventhal syndrome, corticobasal degeneration, argyrophilic grain disease, Guam hirano disease, Tangle-only dementias, the white matter tau lesions with spherical neuroglia inclusion, frontotemporal dementia (for example, FTDP-17), and The chain parkinson's syndrome with chromosome 17.See, for example, Goedert M, Clavaguera F and Tolnay M.The propagation of prion-like protein inclusions in neurodegenerative diseases.Trends Neurol.Sci.In one embodiment, by as described herein anti-at least one measure The one or more of one of body or binding fragment identification tau those anomaly patterns.In some embodiments, measure is IHC. In other embodiments, measure is ELISA.

As used herein, refer to that " specific binding " during antibody means antibody with the antigen more different than on its integrated structure Or the high affinity of epitope combines its target antigen or epitope.

As used herein, term " surface plasma resonance " refers to the protein concentration by detecting biology sensor Medium Culture Change to allow to analyze the optical phenomena of real-time biospecific interaction, such as use BIAcore systems (Pharmacia Biosensor AB,Uppsala,Sweden and Piscataway,N.J.).For other descriptions, referring to the He of embodiment 1 Jonsson, U., wait (1993) Ann.Biol.Clin.51:19-26；Jonsson, U., wait (1991) Biotechniques 11:620-627；Johnsson, B., wait (1995) J.Mol.Recognit.8:125-131；And Johnnson, B., etc. (1991)Anal.Biochem.198:268-277。

" scFv " or " scFv " antibody fragment includes VH the and VL domains of antibody, and wherein these domains are present in list In bar polypeptide chain.Preferably, Fv polypeptides include peptide linker also between VH and VL domains, and it scFv to be formed to think The structure wanted is used for antigen binding.For scFv summary, referring to Pluckthun in The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore are edited, Springer-Verlag, New York, The 269-315 pages (1994).

Term " treatment " as used herein is defined as lacking to the heredity with disease, with disease symptomses or for disease Sunken subject's application or using therapeutic agent, it is therefore an objective to treat, cure, mitigating, alleviating, changing, remedying, improving, improving or shadow Ring disease, the symptom of disease or the genetic defect for disease.In addition, with using the anti-tau antibody of humanization or its tau lacking Symptom during binding fragment composition is compared, if present disclosure composition individually or with another therapeutic agent combined therapy, Cure, mitigate, alleviate, change, remedy, improve, improve or influence treated Alzheimer's or another tau lesions At least one symptom, then it is believed that result is the treatment to potential symptom, controlled regardless of all symptoms of symptom Treat, mitigate, alleviate, change, remedy, improve, improve or influence.

Term " variable " refers to following facts, you can sequence is different extensively between antibody and uses for some parts of variable domain In combination of each specific antibodies to its specific antigen and specificity.However, changeability is in the variable domain of antibody and uneven It is distributed.It, which is enriched in light chain and heavy chain variable domain, is referred to as in three sections of hypervariable region.Variable domain is more highly conserved Part is referred to as framework region (FR).Each self-contained four FR of the variable domain of native heavy and light chain, take beta sheet configuration mostly, lead to Three hypervariable region connections are crossed, the hypervariable region forms annular connection, and forms part beta sheet structure in some cases.Respectively Hypervariable region (HVR) in bar chain is close proximity combined together by FR, with promoting to be formed from the hypervariable region of another chain The antigen binding site of antibody is (referring to Kabat etc., Sequences of Proteins of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health, Bethesda, Md.(1991)).Constant region does not participate in antibodies bind antigen directly, but shows a variety of effector functions, as antibody participates in antibody Dependent cell cytotoxicity (ADCC).

As used herein, term " carrier " is intended to refer to the nucleic acid point for transporting connected another nucleic acid Son.A type of carrier is " plasmid ", and it, which refers to extra DNA section, can be connected into circular double stranded DNA ring therein.It is another The carrier of type is viral vector, wherein extra DNA section can be connected into viral genome.Some carriers can be at them Import autonomous in host cell therein (for example, bacteria carrier and episomal mammalian vectors with bacterial origin of replication) Replicate.Other carriers (for example, non-add type mammalian vector) can be integrated into host cell when being introduced into host cell Genome in, and thus replicated with host genome.In addition, some carriers can instruct the gene being effectively connected with it Expression.Examples of such carriers is referred to herein as " recombinant expression carrier " (or simply, " expression vector ").Usually, recombinant DNA Useful expression vector is often plasmid form in technology.In this manual, " plasmid " and " carrier " is interchangeable, because It is the most frequently used carrier format for plasmid.However, it is contemplated that the other forms including expression vector, such as viral vector (example Such as, replication defect type retrovirus, adenovirus and adeno-associated virus), it exercises identical functions.

It can be will be readily understood that by reference to the detailed description of the embodiment in detail below included herein in the disclosure Hold.Although describing present disclosure by reference to the specific details of its some embodiment, it is not intended to think Such details are the limitations to disclosure scope.

With reference to disease tau humanized antibody

It provided herein is the first man source antibody for human disease/pathologic tau, it can be with conformation dependent Mode identifies tau four different zones.More particularly, humanized antibody as described herein can with they distinguish wild types/ This mode combination QT1-QT4 of the normal tau and tau (disease tau) related to AD conformation it is at least one, two, three It is individual or four.In some embodiments, two, three or four of QT1-QT4 can be occupied by these antibody simultaneously.Change speech It, antibody and tau binding fragment as described herein have following four tau sites any one or more as its " table Position ", some or all of which is comformational epitope：268-HQPGGG-273(SEQ ID NO:148) (at first of Protein tau In repetitive structure domain), 299-HVPGGG-304 (SEQ ID NO:149) (in second repetitive structure domain of Protein tau), 330-HKPGGG-335(SEQ ID NO:150) (in the 3rd repetitive structure domain of Protein tau), and 362-HVPGGG-367 (SEQ ID NO:149) (in the 4th repetitive structure domain of Protein tau).These epitopes herein be also referred to as QT1, QT2, QT3 and QT4.DC8E8 combines whole QT1-QT4；In fact, DC8E8 epitope is HXPGGG, wherein X is any amino Acid.

In one embodiment, antibody and tau binding fragments to Tau 151-139/4R with least 80% it is affine Power, its if the affinity for being no better than parent mouse monoclonal antibody DC8E8 with it as it is good.In one embodiment, Antibody and tau binding fragments retain the specificity of the epitope to the identification of mouse DC8E8 antibody.In one embodiment, these Antibody has one or more favourable biochemical properties (for example, human constant region and the immunogenicity therefore reduced, high expression Level, highly dissoluble, lack obvious albumen aggregation, high stability after purification), it causes them to be best suited for being clinically used for AD and related tau lesions are treated in people.

Rule of thumb optimize DC8E8 humanization by operation framework residue.Initial humanization version RHA/RKA (AX001) 10 times of reductions are shown on the binding affinity to tau relative to chimeric DC8E8 constructs.These data imply one Individual or multiple framework amino acid residues are important for losing humanization DC8E8 with the binding activity seldom thereby resulted in.Phase Instead, single framework reverts mutation is enough to recover the binding affinity in RHD (AX004) humanized antibody.The advantage is not pre- Expect.Other single-point back mutations do not have the unanticipated advantage of identical on binding affinity.See, for example, RHF/RKA (AX006) and RHG/RKA (AX007) construct.And next optimum antibody carries 10 back mutations.Referring to AX002.In all back mutations combination tested, AX004 proves there is best combination affinity to tau (in this paper institutes In the measure stated), although there is single back mutation, and unlike other single back mutation constructs.What affinity improved Other humanized constructs include such as AX002, AX005, AX037 and AX014, AX016, AX017, AX038, respectively in RKA In RKB light chain versions.

There is also provided the tau binding fragments (such as antibody moiety) of humanized antibody as described herein.These fragments Can by they distinguish wild types/normal tau and tau related to AD conformation it is this in a manner of with reference to QT1-QT4 at least One, two, three or four (defined above).In some embodiments, all four of QT1-QT4 can be by this paper institutes The antibody stated or four of tau binding fragments occupy.In some embodiments, fragment or part with mouse monoclonal antibody DC8E8 identicals affinity and property combination tau.For example, can combine one or more of QT1-QT4 antibody fragment or Part include, but are not limited to Fab (for example, passing through papain digestion), Fd, Fab'(for example, by pepsin digestion and Partial reduction) and present invention offer F (ab')₂(for example, passing through pepsin digestion), facb are (for example, pass through fibrin Lyase digests), pFc'(is for example, by pepsin or plasmin digestion), Fd (for example, by pepsin digestion, Partial reduction and reassociate), Fv or scFv (for example, passing through Protocols in Molecular Biology) fragment.Referring also to William E.Paul (editor) Fundamental Immunology, the 6th edition, Lippincott Williams＆Wilkins, NY, N.Y.2008, with It is incorporated herein entirely through reference.Any other fragment of half-life period has been added on other molecules by being conjugated to, has been wrapped The fragment for including Pegylation is also within the scope of the invention.Can by it is such as conventionally known in the art or provided herein is enzyme Cutting, synthesis or recombinant technique produce some fragments.Antibody gene can also be used, and (wherein one or more terminator codons are Through introducing natural stop site upstream) produce the antibody of a variety of clipped forms.For example, coding F (ab') can be designed₂Heavy chain portion The combination gene divided is with CH1 domains and/or the DNA sequence dna of hinge area including encoding heavy chain.It can be used by routine techniques Chemical method links together some of antibody, or can be prepared as abutting egg using conventional genetic renovation technique In vain.

According to the amino acid sequence of the constant domain of their heavy chain, complete antibody can be assigned as different " species ". In one embodiment, there is the complete antibody of six main species：IgA, IgD, IgE, IgG, IgY and IgM, and these In it is several can be further separated into " subclass " (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.It is corresponding α (α), δ (δ), ε (ε), γ (γ) and μ (μ) are referred to as in the heavy-chain constant domains of different types of antibody.It is different types of to exempt from The subunit structure and 3-d modelling of epidemic disease globulin are well-known.In one embodiment, antibody as described herein has The constant region of any existing immunogene isotype.For example, constant region can be λ or κ areas and γ -1, γ -2, γ -3, or the areas of γ 4 Constant region.The constant region of isotype is mixed also scope of the present disclosure interior (such as being mixed with IgG4 IgG1). Recognize that some isotypes are better than other isotypes.Bruggemann M,Williams GT,Bindon CI,Clark MR, Walker MR,Jefferis R,Waldmann H,Neuberger MS(1987)Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies.J.Exp.Med.166,1351-1361；Bindon CI,Hale G,Bruggemann M,Waldmann H (1988)Human monoclonal IgG isotypes differ incomplement activating function at the level of C4 as well as C1q.J.Exp.Med.168,127-42；Shaw DR,Khazaeli MB, LoBuglio AF(1988)Mouse/human chimeric antibodies to a tumor associated antigen:biologic activity of the four human IgG subclasses.J.Natl.Cancer Inst.80,1553-9；Steplewski Z,Sun LK,Shearman CW,Ghrayeb J,Daddona P,Koprowski, H(1988)Biological activity of human-mouse IgG1,IgG2,IgG3,and IgG4chimeric monoclonal antibodies with antitumor specificity.Proc.Natl.Acad.Sci.USA 85, 4852-6.In one group of experiment, as described in these bibliography, comparing people's isotype IgM, IgG1, IgG2, IgG3, (two same It is kind special-shaped), IgG4, IgA and IgE cracking of autologous complement mediation and the cytotoxicity of antibody dependent cellular mediation (ADCC).Cracking for complement-mediated, people IgM and IgG1 are proved to be maximally effective, and IgG3 two allografts are second Best.IgG2 produces weak cracking, and other isotypes seem not produce cracking.ADCC result is that IgG1 is followed closely IgG2, IgG3 or IgG4 are highly effective again, and this depends on measure (for example, cell type).Other isotypes, including IgM is Invalid.But antibody has many other activity (for example, by macrophage promote opsonic action), and be finally difficult to or It may even not predict which kind of isotype will have optimal properties set for their intended use.

Another factor for influenceing the immunogenicity of humanized antibody is polymorphic determinants in constant region be present.Can be by this A little differences minimize the antigenicity for reduction.It was observed that human IgG has 18 allografts, it has rational in colony Frequency：IgG1 has 4；IgG2 has 1；IgG3 has 13；IgG4 has 0.WHO(1976)Review of the notation for the allotypic and related markers of human immunoglobulins.Eur.J.Immunol.6,599-601.Additionally, there are three allografts of people's k light chains.Some are same Kind abnormal shape is present in some individuals, and is not present in other.Some are mainly expressed in Japanese population.

Many methods for humanizing non-human antibodies have been described in the art.In one embodiment, people Source antibody, which has from non-people source, introduces one of those or more amino acid residue.These non-human amino acid residues often claim For " input " residue, it is normally taken from " inputting " variable domain.In one embodiment, can be essentially according to Winter and its colleague (Jones etc., Nature, 321:522-525(1986)；Riechmann etc., Nature, 332:323-327(1988)； Verhoeyen etc., Science, 239:1534-1536 (1988)) method, by by hypervariable region sequence replace human antibody phase Sequence is answered to carry out humanization.Therefore, such " humanization " antibody is chimeric antibody (U.S. Patent number 4,816,567), wherein Substantially less than whole person's variable domain is replaced by the corresponding sequence of non-human species.In some embodiments, humanization resists Body is human antibody, and some of some hypervariable region residues are replaced with some possible FR residues by the residue in similar site in rodent antibodies Change.

It is important to reducing antigenicity that selection people's variable domain (light chain and heavy chain), which is ready to use in and prepares humanized antibody,.According to So-called " best fit " method, for the sequence of the whole library screening rodent antibodies variable domain of known people's variable domain sequence Row.With the most similar human sequence's (complete variable region or only framework) of sequence of rodent and then being accepted as people's framework region (FR) For humanized antibody (Sims etc., J.Immunol., 151:2296(1993)；Chothia etc., J.Mol.Biol., 196:901 (1987)).Another method uses specific framework region, and it is from the shared of all human antibodies of light chain or the specific subgroup of heavy chain Sequence.Identical framework can be used for some different humanized antibodies (Carter etc., Proc.Natl.Acad.Sci.USA, 89: 4285(1992)；Presta etc., J.Immunol, 151:2623(1993)).

In some embodiments, it is important that retain to the high-affinity of antigen and parent's (for example, mouse antibodies) Other advantageous biological property humanized antibodies.In order to realize the target, according to an embodiment, by analyzing parental array With the process of a variety of notional humanization products, humanized antibody is prepared using the threedimensional model of parent and humanized sequence. Three dimensional immunoglobulin model can be generally obtained, and it is known to those skilled in the art.Computer program can be obtained, its Illustrate and show the three-dimensional conformation structure of selected candidate immunoglobulin sequences sequence.The inspection of these displayings allows analysis residue to exist Possibility effect in candidate immunoglobulin sequences functional nucleotide sequence, i.e. analyzing influence candidate immunoglobulin sequences combine the ability of its antigen Residue.So, it can be selected from acceptor and list entries and combine FR residues, it is such as right to realize desired antibody characteristic The affinity that target antigen improves.In general, directly and most substantially participation influences antigen binding to some hypervariable region residues.

Table 2

Previous table 2 provides the SEQ ID NO of some amino acid sequences as described herein.In another embodiment, As described herein humanized antibody or tau binding fragments include SEQ ID NO.1,2,3 respectively as heavy chain CDR1,2 and 3, SEQ ID NO.4,5,6 are respectively as light chain CDR 1,2 and 3.In some embodiments, the antibody or fragment include at least one Individual CDR, is such as defined according to Kabat, and its sequence after SEQ ID NO.1-6 any one progress optimal comparison with having at least 80%, the homogeneity of preferably at least 85%, 90%, 95% and 98%.

In one embodiment, antibody (AX001) or its tau binding fragment include weight chain variable district RHA (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.13.IgG1 (AX001-IgG1) or IgG4 are provided for the antibody (AXON001-IgG4) constant region.

In one embodiment, antibody (AX002) or its tau binding fragment include weight chain variable district RHB (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.14.IgG1 (AX002-IgG1) or IgG4 are provided for the antibody (AXON002-IgG4) constant region.

In one embodiment, antibody (AX003) or its tau binding fragment include weight chain variable district RHC (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.15.IgG1 (AX003-IgG1) or IgG4 are provided for the antibody (AXON003-IgG4) constant region.

In one embodiment, antibody (AX004) or its tau binding fragment include weight chain variable district RHD (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.16.IgG1 (AX004-IgG1) or IgG4 are provided for the antibody (AXON004-IgG4) constant region.

In one embodiment, antibody (AX005) or its tau binding fragment include weight chain variable district RHE (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.17.IgG1 (AX005-IgG1) or IgG4 are provided for the antibody (AXON005-IgG4) constant region.

In one embodiment, antibody (AX006) or its tau binding fragment include weight chain variable district RHF (SEQ ID ) and light chain variable district RKA (SEQ ID NO.27) NO.18.IgG1 (AX006-IgG1) or IgG4 are provided for the antibody (AXON006-IgG4) constant region.

In one embodiment, antibody (AX007) or its tau binding fragment include weight chain variable district RHG (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.19.IgG1 (AX007-IgG1) or IgG4 are provided for the antibody (AXON007-IgG4) constant region.

In one embodiment, antibody (AX008) or its tau binding fragment include weight chain variable district RHH (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.20.IgG1 (AX008-IgG1) or IgG4 are provided for the antibody (AXON008-IgG4) constant region.

In one embodiment, antibody (AX009) or its tau binding fragment include weight chain variable district RHI (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.21.IgG1 (AX009-IgG1) or IgG4 are provided for the antibody (AXON009-IgG4) constant region.

In one embodiment, antibody (AX010) or its tau binding fragment include weight chain variable district RHJ (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.22.IgG1 (AX010-IgG1) or IgG4 are provided for the antibody (AXON010-IgG4) constant region.

In one embodiment, antibody (AX011) or its tau binding fragment include weight chain variable district RHK (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.23.IgG1 (AX011-IgG1) or IgG4 are provided for the antibody (AXON011-IgG4) constant region.

In one embodiment, antibody (AX012) or its tau binding fragment include weight chain variable district RHL (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.24.IgG1 (AX012-IgG1) or IgG4 are provided for the antibody (AXON012-IgG4) constant region.

In one embodiment, antibody (AX013) or its tau binding fragment include weight chain variable district RHA (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.1.IgG1 (AX013-IgG1) or IgG4 are provided for the antibody (AXON013-IgG4) constant region.

In one embodiment, antibody (AX014) or its tau binding fragment include weight chain variable district RHB (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.2.IgG1 (AX014-IgG1) or IgG4 are provided for the antibody (AXON014-IgG4) constant region.

In one embodiment, antibody (AX015) or its tau binding fragment include weight chain variable district RHC (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.15.IgG1 (AX015-IgG1) or IgG4 are provided for the antibody (AXON015-IgG4) constant region.

In one embodiment, antibody (AX016) or its tau binding fragment include weight chain variable district RHD (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.16.IgG1 (AX016-IgG1) or IgG4 are provided for the antibody (AXON016-IgG4) constant region.

In one embodiment, antibody (AX017) or its tau binding fragment include weight chain variable district RHE (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.17.IgG1 (AX017-IgG1) or IgG4 are provided for the antibody (AXON017-IgG4) constant region.

In one embodiment, antibody (AX018) or its tau binding fragment include weight chain variable district RHF (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.18.IgG1 (AX018-IgG1) or IgG4 are provided for the antibody (AXON018-IgG4) constant region.

In one embodiment, antibody (AX019) or its tau binding fragment include weight chain variable district RHG (SEQ ID ) and light chain variable district RKA (SEQ ID NO.27) NO.19.IgG1 (AX019-IgG1) or IgG4 are provided for the antibody (AXON019-IgG4) constant region.

In one embodiment, antibody (AX020) or its tau binding fragment include weight chain variable district RHH (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.20.IgG1 (AX020-IgG1) or IgG4 are provided for the antibody (AXON020-IgG4) constant region.

In one embodiment, antibody (AX021) or its tau binding fragment include weight chain variable district RHI (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.21.IgG1 (AX021-IgG1) or IgG4 are provided for the antibody (AXON021-IgG4) constant region.

In one embodiment, antibody (AX022) or its tau binding fragment include weight chain variable district RHJ (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.22.IgG1 (AX022-IgG1) or IgG4 are provided for the antibody (AXON022-IgG4) constant region.

In one embodiment, antibody (AX023) or its tau binding fragment include weight chain variable district RHK (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.23.IgG1 (AX023-IgG1) or IgG4 are provided for the antibody (AXON023-IgG4) constant region.

In one embodiment, antibody (AX024) or its tau binding fragment include weight chain variable district RHL (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO.24.IgG1 (AX024-IgG1) or IgG4 are provided for the antibody (AXON024-IgG4) constant region.

In one embodiment, antibody (AX025) or its tau binding fragment include weight chain variable district RHA (SEQ ID NO.13) and light chain variable district VK (SEQ ID NO.8)).IgG1 (AX025-IgG1) or IgG4 are provided for the antibody (AXON025-IgG4) constant region.

In one embodiment, antibody (AX026) or its tau binding fragment include weight chain variable district RHB (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.14.IgG1 (AX026-IgG1) or IgG4 (AXON026- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX027) or its tau binding fragment include weight chain variable district RHC (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.15.IgG1 (AX027-IgG1) or IgG4 (AXON027- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX028) or its tau binding fragment include weight chain variable district RHD (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.16.IgG1 (AX028-IgG1) or IgG4 (AXON028- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX029) or its tau binding fragment include weight chain variable district RHE (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.17.IgG1 (AX029-IgG1) or IgG4 (AXON029- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX030) or its tau binding fragment include weight chain variable district RHF (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.18.IgG1 (AX030-IgG1) or IgG4 (AXON030- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX031) or its tau binding fragment include weight chain variable district RHG (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.19.IgG1 (AX031-IgG1) or IgG4 (AXON031- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX032) or its tau binding fragment include weight chain variable district RHH (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.20.IgG1 (AX032-IgG1) or IgG4 (AXON032- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX033) or its tau binding fragment include weight chain variable district RHI (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.21.IgG1 (AX033-IgG1) or IgG4 (AXON033- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX034) or its tau binding fragment include weight chain variable district RHJ (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.22.IgG1 (AX034-IgG1) or IgG4 (AXON034- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX035) or its tau binding fragment include weight chain variable district RHK (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.23.IgG1 (AX035-IgG1) or IgG4 (AXON035- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX036) or its tau binding fragment include weight chain variable district RHL (SEQ ID ) and light chain variable district VK (SEQ ID NO.8) NO.24.IgG1 (AX036-IgG1) or IgG4 (AXON036- are provided for the antibody IgG4) constant region.

In one embodiment, antibody (AX037) or its tau binding fragment include weight chain variable district RHE (SEQ ID ) and light chain variable district RKA (SEQ ID NO.26) NO.25.IgG1 (AX037-IgG1) or IgG4 are provided for the antibody (AXON037-IgG4) constant region.

In one embodiment, antibody (AX038) or its tau binding fragment include weight chain variable district RHM (SEQ ID ) and light chain variable district RKB (SEQ ID NO.27) NO..IgG1 (AX037-IgG1) or IgG4 (AXON037- are provided for the antibody IgG4) constant region.

In another embodiment, the present invention provide heavy chain of antibody, its include selected from weight chain variable district RHA, RHB, RHC, Any one variable region of RHD, RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL and RHM.These any variable regions can connect Take over the constant region of who isotype, including the constant region with mixing isotype.

In another embodiment, the present invention provides the antibody light chain for including the variable region selected from RKA and RKB.It is any this A little variable regions can connect the constant region of anyone isotype, including the constant region with mixing isotype.

In another embodiment, the present invention provides the heavy chain of antibody of SEQ ID NO.28-40 and 43-55 any one.

In another embodiment, the present invention provides the antibody light chain selected from SEQ ID NO.57-59.

The heavy chain and light chain variable district of humanized antibody can connect at least some people constant region.Such as above in definition Described, the selected section of constant region depends on whether that the cytotoxicity of desired antibody dependent cellular mediation, antibody dependent are thin The phagocytosis of born of the same parents and/or complement-dependent cytotoxicity.In one embodiment, people's isotype (isotopes) IgG1 and IgG3 With complement-dependent cytotoxicity, and people's isotype IgG2 and IgG4 do not have.In one embodiment, human IgG1 and IgG3 also induction ratio human IgG2 and the stronger cell-mediated effector functions of IgG4.Constant region of light chain can be λ or κ.It is exemplary People's light chain κ constant regions have SEQ ID NO:170 amino acid sequence.SEQ ID NO can be omitted:170 N-terminal essence ammonia Acid, in said case light chain κ constant regions there is SEQ ID NO:171 amino acid sequence.Exemplary human IgG1's light chain constant Area has SEQ ID NO:172 amino acid sequence.The exemplary heavy chain constant region of human IgG 4 has SEQ ID NO:173 amino Acid sequence.Can by antibody expression be the tetramer containing two light chains and two heavy chains, single heavy chain, light chain, Fab, Fab', F (ab') 2 and Fv or single-chain antibody, wherein heavy chain are connected with light chain maturation variable domain by sept.

The cDNA sequence of the constant region of encoding human antibody is known to those of ordinary skill in the art.In an embodiment In, it is as follows for example, by exemplary cDNA sequence obtained by GenBank (being each incorporated to it entirely through reference)：Human IgG1 Constant heavy area：GenBank accession number:J00228；Human IgG2's constant heavy area：GenBank accession number:J00230；Human IgG 3 Constant heavy area：GenBank accession number:X04646；The constant heavy area of human IgG 4：GenBank accession number:K01316；It is light with people κ Chain constant region：GenBank accession number:J00241.In one embodiment, can further be modified according to known method constant Area.For example, in IgG4 constant regions, residue S241 can be mutated into proline (P) residue to allow complete two at hinge Sulfide linkage is formed (see, for example, Angel etc., Mol.Immunol.1993；30:105-8).

Multiple variable regions corresponding to some humanized antibodies as described herein are outlined in order to become apparent from, in table 3 below A plurality of amino acid sequence.

Table 3

Table 4 below outlines the amino acid sequence of a plurality of full length sequence corresponding to mouse as described herein and humanized antibody Row.

Table 4

Table 5 below outlines a plurality of amino of a plurality of full length sequence of the light chain corresponding to humanized antibody as described herein Acid sequence.

The constant K of complete humanization variable light	SEQ ID NO.
		RKA	57
RKB	58
		cDC8E8κ	59

Table 5

There is also provided chimeric antibody and its tau binding fragments.In one embodiment, chimeric antibody or its tau knots Close fragment include weight chain variable district DC8E8 VH (SEQ ID NO.9) and light chain variable district DC8E8 VK (SEQ ID NO.10) and Human IgG1's constant region (SEQ ID NO.172) is used for heavy chain together and κ constant regions (SEQ ID NO.170) are used for light chain.Another In one embodiment, chimeric antibody or its tau binding fragment include weight chain variable district DC8E8 VH (SEQ ID NO.9) and light chain Variable region DC8E8 VK (SEQ ID NO.10) are used for heavy chain together with the constant region of human IgG 4 (SEQ ID NO.173) and κ is constant (SEQ ID NO.170) is used for light chain in area.

In one embodiment, humanized antibody or its tau binding fragments/part are prepared by recombinant.In another embodiment party In case, humanized antibody or its tau binding fragments/part are prepared at least partially by chemical synthesis.In one embodiment, It is prepared by recombinant chimeric antibody or its tau binding fragments/part.In another embodiment, at least partially by chemical synthesis system Standby chimeric antibody or its tau binding fragments/part.

Table 6 outlines the sequence of some other humanization correlation molecules as described herein.

Table 6

Cover the amino acid sequence modifications of antibody as described herein.For example, it is desirable to improve antibody binding affinity and/or Other biological property.The amino acid sequence variation of antibody is incorporated into antibody nucleic acids by the way that appropriate nucleotides is changed, or Prepared by peptide symthesis.It is such modification include for example, in the amino acid sequence of antibody residue missing, and/or insertion and/ Or substitution.Any combinations for being lacked, being inserted and being substituted are to realize final construct, as long as final construct possesses the phase The feature of prestige.Amino acid change can also change process after the translation of antibody, such as change quantity or the position of glycosylation site.

What present disclosure also included is antibody and binding fragment, and its sequence is changed by following, wherein passing through Will be at least one, especially at least two, more particularly at least 3 or more conservative replacements are introduced between SEQ ID NO: 13-25 and SEQ ID NO:In 26 and 27 sequence, so that the antibody substantially maintains its repertoire.

Some amino acid from people's maturation variable framework residues can be selected to be used to substitute, this is based on it to CDR structures As and/or with reference to antigen, the interaction between heavy chain and light chain is mediated, the interaction with constant region, is turned into for it is expected Or unexpected posttranslational modification site, turn into people's variable region sequences uncommon residue on its position and thus may Possibility influence with immunogenicity etc. reason.The those of ordinary skill of this area one will know how some amino acid of picking For substituting, the substituted result is then assessed.In many embodiments, rule of thumb select for substituting in framework Position and amino acid to be replaced.

49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can also be carried out in CDR.A kind of possible variation is utilized from the corresponding residual of people's CDR sequence Base, substitute mouse DC8E8 typical from the CDR of the people's acceptor sequence for design example humanized antibody corresponding residue Some residues in the CDR of antibody.In some antibody, only part CDR, i.e., (it is referred to as with reference to the subgroup of required CDR residues SDR) need to retain in humanized antibody to combine.Can be based on previous research, from outside Chothia hypervariable loops Kabat CDR regions (Chothia, J.Mol.Biol.196:901,1987), by molecule modeling and/or rule of thumb, or such as Gonzales etc., Mol.Immunol.41:Identified described in 863 (2004) and do not contact antigen and the CDR residues not in SDR (such as the residue H60-H65 in CDR H2 is often unwanted).It is one or more wherein in such humanized antibody On the position that donor CDR residue deletions or wherein complete donor CDR are omitted, the amino acid that occupies the position can be occupy by The amino acid of relevant position (being numbered by Kabat) in body antibody sequence.This receptor that CDR includes takes to donor amino acid Algebraic quantity reflects the balance that competition considers.Such be substituted in is reduced the quantity of mouse amino acid in humanized antibody and therefore dropped May be favourable in low possible immunogenicity.However, substitution can also cause affinity to change, and preferably avoid affinity It is obvious to reduce.The position for substituting in CDR and amino acid to be replaced can also rule of thumb be selected.

In one embodiment, antibody and its tau binding fragments include weight chain variable district, and it includes SEQ ID respectively NO 1,2 and 3 CDR-H1, CDR-H2 and CDR-H3, and with SEQ ID NO.28-40 (complete RHA-RHM) ripe heavy chain With at least 90% homogeneity；And light chain variable district, its respectively comprising SEQ ID NO.4,5 and 6 CDR-L1, CDR-L2 and CDR-L3, and it is same with least 90% with SEQ ID NO.57 (RKA) or SEQ ID NO.58 (RKB) ripe light chain Property.In some embodiments, ripe weight chain variable district and SEQ ID NO.28-40 any one have at least 95%, 96%th, 97%, 98% or 99% homogeneity.In some embodiments, ripe light chain variable district and SEQ ID NO.57 or SEQ ID NO.58 any one has at least 95%, 96%, 97%, 98% or 99% homogeneity.

For identifying that some residues of antibody or the process useful of region (it is the optimum position for mutagenesis) are referred to as " third Propylhomoserin scanning mutagenesis ", it is such as Cunningham and Wells Science, and 244:1081-1085 (1989) is described.Herein, identify Residue or target residues group (for example, electrically charged residue, such as arg, asp, his, lys and glu) and by neutral or negatively charged Amino acid (most preferably alanine or polyalanine) is replaced to influence the interaction of amino acid and antigen.Then by substituting Refined those the amino acid positions that function sensitive is shown to substitution of further or other variants are introduced on site or for substitution site Put.Therefore, although the site for introducing variant amino acid sequence is predetermined, emergent properties need not make a reservation in itself. For example, in order to analyze to the mutation performance on anchor point, Alanine-scanning is carried out on target codon or region or is lured at random Become and screen the expectation activity of expressed antibody variants.

Amino acid sequence insertion includes length in the range of a residue to the polypeptide for containing hundreds of or more residues Amino and/or carboxyl-terminal fusion, and inserted between the sequence of single or multiple amino acid residues.The example bag of end insertion Include the antibody of antibody or another therapeutic agent of fusion with N- terminal methionyl residues.Other insertion variant bags of antibody molecule N- the or C- ends of fusion antibody are included to enzyme (for example, for ADEPT) or the polypeptide for the serum half-life for increasing antibody.

Another type of variant is amino acid substitution variant.These variants at least one amino acid in antibody molecule is residual Base is replaced by different residues.In one embodiment, it is most interested in for substituting the site of mutagenesis to include hypervariable region, but also contains FR is covered to change.Above and hereafter conservative replacement is shown in table.The more realities named in amino acid classes can be introduced into Matter changes and screens product.

By selecting to realize antibody biological property to substitution dramatically different in maintaining the influence of following aspect at it Essence modification：(a) substitute the structure of polypeptide backbone in area, such as be used as folding or helical conformation, molecule on (b) target site Electric charge or hydrophobicity, or (c) side-chain bulk.Amino acid can be grouped according to the similitude of its side chain properties (A.L.Lehninger, Biochemistry, second edition, the 73-75 pages, Worth Publishers, New York (1975)):

(1) it is nonpolar：Ala(A)、Val(V)、Leu(L)、Ile(I)、Pro(P)、Phe(F)、Trp(W)、Met(M)

(2) uncharged polarity：Gly(G)、Ser(S)、Thr(T)、Cys(C)、Tyr(Y)、Asn(N)、Gln(Q)

(3) it is acid：Asp(D)、Glu(E)

(4) it is alkaline：Lys(K)、Arg(R)、His(H)

Or the residue naturally occurred can be grouped based on other common side chain properties：

(1) it is hydrophobic：Norleucine, Met, Ala, Val, Leu, Ile

(2) neutral hydrophilic：Cys、Ser、Thr、Asn、Gln；

(3) it is acid：Asp、Glu；

(4) it is alkaline：His、Lys、Arg；

(5) residue of chain orientation is influenceed：Gly、Pro；

(6) aromatic series：Trp、Tyr、Phe.

Non-conservation substitution generally causes the member that the member of one of these species is exchanged into another species to turn into required.

Typically can also utilize serine substitution be not involved in maintain antibody correct conformation any cysteine residues with Carry high molecular antioxidative stabilizer and prevent abnormal crosslinking.On the contrary, it is steady to improve its to add cysteine key to antibody Qualitative (particularly wherein antibody is antibody fragment, during such as Fv fragments).

Particularly preferred substitution variant type includes one or more of substitution parental antibody (such as humanization or human antibody) Individual some hypervariable region residues.Usually, change for the selected gained variant of further exploitation relative to producing its parental antibody and will have Kind biological property.For producing affinity maturation of the usual manner of such substitution variant including the use of phage display. Substituted in short, some hypervariable region sites (such as 6-7 site) are mutated with producing all possible amino on each site. Therefore antibody variants caused by are shown as from filamentous phage particle as the gene III product packed with M13 in each particle The monovalent fashion of fusion.Then its biological activity as disclosed herein of the variant of screening phage display is (for example, with reference to affine Power).In order to identify the candidate hypervariable region site for modification, alanine scanning mutagenesis can be carried out and mainly promote antigen to identify With reference to some hypervariable region residues.Or or in addition, it may be beneficial to analyze the crystal structure of antigen-antibody complexes to identify Contact point between antibody and people tau.Such contact residues and neighbouring residue are for being entered according to the technology described in detail herein The candidate of row substitution.Once generating such variant, make one group of variant be subjected to screening as described herein and can select one The antibody with advantageous property is used to further develop in individual or multiple related assays.

The amino acid variant of another type of antibody changes the initial glycosylation pattern of antibody.Missing is represented by changing The one or more glycosyls being not present in the one or more carbohydrate portions found in antibody, and/or addition antibody Change site.

The glycosylation of antibody is usually N- connections or O- connections.The finger carbohydrate portions of N- connections and asparagus fern acyl The connection of the side chain of amine residue.Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, wherein X are except dried meat ammonia Any amino acid outside acid, it is the recognition sequence of the enzyme connection of carbohydrate portions and asparagine side chain.Therefore, these The potential glycosylation site of generation in polypeptide be present in any of tripeptide sequence.The glycosylation of O- connections refers to sugared N- acetyl gala One of osamine, galactolipin or xylose are connected to hydroxy-amino-acid, most commonly on serine or threonine, although can also make With 5- hydroxy-prolines or 5- oxylysines.

Advantageously complete to add glycosylation site to antibody so that it contains one or more by changing amino acid sequence Above-mentioned tripeptide sequence (for the glycosylation site of N- connections).Can also be by adding one or more in the sequence to initial antibodies Individual serine or threonine residues are changed (for O- connections by one or more serines or threonine residues substitution Glycosylation site).

When wherein antibody includes Fc areas, thus it is possible to vary adhere to carbohydrate thereon.For example, in US Pat Appl No US 2003/0157108 A1, Presta, L. referring also to the A1 of US 2004/0693621 (Kyowa Hakko Kogyo Co., Ltd the antibody with ripe carbohydrate structure is described in), it lacks the trehalose being attached in the Fc areas of antibody. Following antibody are with reference in WO03/011878, Jean-Mairet etc. and U.S. Patent number 6,602,684, Umana etc., its carbon water N-Acetyl-D-glucosamine (GlcNAc) is halved in compound to be attached in the Fc areas of antibody.In WO97/30087, Patel etc. The WO98/58964 (Raju, S.) and WO99/22764 of the antibody in its Fc area are attached to referring to the carbohydrate on change Following antibody are reported in (Raju, S.), at least one galactose residue is attached in the Fc areas of antibody in its oligosaccharides.

It is expected the antibody of modification present disclosure or the half-life period of tau binding fragments.In one embodiment, by one Or multiple Fc amino acid mutations to be to increase the half-life period of antibodies in blood, or Fc one or more sugared portions are wherein lacked Point, or one or more sugar moieties are added to increase the blood halflife of antibody.Common plasma protein, such as human serum albumins (HSA) humanized antibody display long half-life period and immunoglobulin (Igs), is included, typically 2-3 weeks, this was attributable to it Interacted with the specificity of neonatal Fc receptor (FcRn), it causes inclusion body to recycle (Ghetie (2002) Immunol Res,25:97-113).On the contrary, most of other pharmaceutically albumen interested, particularly recombinant antibody fragment, hormones and dry Disturb element and be subjected to quick (blood) removing.This is especially true below about the protein of 70kDa kidney filtering thresholds to size (Caliceti(2003)Adv Drug Deliv Rev 55:1261-1277).In these cases, unmodified pharmaceutical protein Plasma half-life be significantly less than 1 hour.This can limit its use in most for the treatment of uses.It is lasting in order to realize Pharmacological action, the patient being also improved adapt to -- required spacing of doses extends to some days or even some weeks -- in this area Have built up and describe some strategies and be used for biopharmacy drug development.

In other embodiments, can by Pegylation or other be conjugated on other aggregations modified antibodies or Its tau binding fragment is to influence half-life period or circulation time.Polymer can be any molecular weight, and can be branch or Non- branch.For polyethylene glycol, (term " about " represents in polyethylene glycol preferred molecular weight between about 1kDa- about 100kDa Preparation in, some molecules will weigh more than the molecular weight, and some are less) in order to operating and prepare.It can be used His size, this depend on desired treatment compose (for example, it is desirable to duration of sustained release, (if any) to biology Activity influence, operation facility, antigenic degree or lack with polyethylene glycol to treatment albumen or the like other Know influence).For example, polyethylene glycol can have about 200,500,1000,1500,2000,2500,3000,3500,4000, 4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10,000、10,500、11,000、 11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、 17,000、17,500、18,000、18,500、19,000、19,500、20,000、25,000、30,000、35,000、40,000、 50,000th, 55,000,60,000,65,000,70,000,75,000,80,000,85,000,90,000,95,000 or 100, 000Da mean molecule quantity.Such as in U.S. Patent number 5,643,575；Morpurgo etc., Appl.Biochem.Biotechnol.56:59-72(1996)；Vorobjev etc., Nucleosides Nucleotides 18: 2745-2750(1999)；With Caliceti etc., Bioconjug.Chem.10:The poly- of branch is described in 638-646 (1999) Ethylene glycol.Polyethylene glycol can be attached on protein by the connection of any residue with many amino acid residues.It is for example, poly- Ethylene glycol can pass through the covalent bond connecting peptides with lysine, histidine, aspartic acid, glutamic acid or cysteine residues. One or more reactive chemistries can be used for polyethylene glycol being attached to specific amino acid residue (for example, lysine, group ammonia Acid, aspartic acid, glutamic acid or cysteine) on or more than one type amino acid residue (for example, lysine, histidine, Aspartic acid, glutamic acid, cysteine and combinations thereof) on.

Or antibody or its fragment can pass through and albumin (including but not limited to recombination human serum albumin or its piece Section or variant (see, for example, distribution in U.S. Patent number on March 2nd, 5,876,969,1999, EP Patent 0 413 622, and U.S. Patent number distribution on June 16th, 5,766,883,1998, is incorporated herein with it entirely through reference)) or other circulatings Liquid eggs white matter such as transferrins or ferritin and there is increased Half-life in vivo.In one embodiment, it is of the invention Polypeptide and/or antibody (including its fragment or variant) (that is, EP patents 0 322 are merged with the mature form of human serum albumins The amino acid/11-585 of the human serum albumins shown in 094 Fig. 1 and 2), it is incorporated herein with it entirely through reference.The present invention It is also covered by the polynucleotides of the encoding fusion protein of present disclosure.

Can also chemical modification antibody or its tau binding fragment to provide extra advantage, as polypeptide it is increased it is soluble, Stability and circulation time (Half-life in vivo) or the immunogenicity reduced (see, for example, U.S. Patent number 4,179,337).Can With from water-soluble polymer such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, glucan, polyvinyl alcohol etc. Select to be used for derivative chemical part.Can in the random site of intramolecular, or intramolecular precalculated position modified antibodies and Its tau binding fragment and its chemical part that can adhere to including one, two, three or more.

It is expected to modify the antibody or tau binding fragments its effector functions of present disclosure, such as to strengthen the antigen of antibody The cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of dependent cell mediation.This can be by antibody One or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors are introduced in Fc areas to realize.Or or extraly, Ke Fc areas introduce cysteine residues, by This allows to form interchain disulfide bond in this region.Therefore generation same diabodies can have improve internalization capability and/ Or the cell killing and antibody-dependent cytotoxicity (ADCC) of increased complement-mediated.Referring to Caron etc., J.Exp Med.176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922(1992).Or it can transform anti- Body, its complement lysis and ADCC abilities with binary Fc areas and thus with enhancing.Referring to Anti- such as Stevenson Cancer Drug Design 3:219-230(1989).

WO00/42072 (Presta, L.), which describes, has improved ADCC functions in the case where human effector cell be present Antibody, wherein including 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in the antibody Qi Fc areas.Preferably, there is improved ADCC antibody in Fc areas Position 298,333 and/or 334 at comprising substitution.Preferably, the Fc areas of change are human IgG1 Fc areas, and it includes these positions On one, two or three opening position substitution or be made from it.

C1q with change is combined and/or the antibody of complement-dependent cytotoxicity (CDC) be described in WO99/51642, U.S. Patent number 6,194,551B1, U.S. Patent number 6,242,195B1, U.S. Patent number 6,528,624B1 and U.S. Patent number In 6,538,124 (Idusogie etc.).The amino acid position 270,322,326,327,329,313,333 in antibody Qi Fc areas And/or 334 one or more on include 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor., can be by salvage receptor in order to increase the serum half-life of antibody It is incorporated into reference to epitope as in the antibody (especially antibody fragment) described in U.S. Patent number 5,739,277.As used herein, Term " salvage receptor binding epitope " refer to be responsible for increase IgG molecules inside serum half-life IgG molecules (for example, IgG1, IgG2, IgG3 or IgG4) Fc areas epitope.

Described in WO00/42072 (Presta, L.) with neonatal Fc receptor (FcRn) improve combine and it is increased The antibody of half-life period.These antibody include the Fc areas wherein with one or more substitutions, and it improves Fc areas and FcRn combination. For example, Fc areas can position 238,256,265,272,286,303,305,307,311,312,317,340,356,360, 362nd, there is substitution at 376,378,380,382,413,424 or 434 one or more.It is preferable that there is the FcRn knots improved Amino acid is included at the one, two or three of the position 307,380 and 434 in the antibody variants Qi Fc areas comprising Fc areas closed Substitution.

In one embodiment, humanized antibody or its tau binding fragment/be partly monoclonal.It is as described herein Monoclonal antibody (mAbs) is to be obtained from the antibody of the substantially group of homogeneous antibody, i.e., each antibody comprising group is except possible Can be identical with a small amount of existing naturally undergo mutation outer.Monoclonal antibody is high special, and it is for single anti- Point in situ.Each mAb is for the single determinant (epitope) on antigen.Except its specificity, monoclonal antibody be it is favourable, because It can be synthesized, not polluted by other immunoglobulins by hybridoma culture for them.Modifier " monoclonal " represents antibody certainly The characteristic that substantially antibody population of homogeneity obtains, and should not be construed as requiring to produce antibody by any ad hoc approach.Example Such as, it can prepare, or can be treated by recombinant DNA method preparation used according to the invention in immortal B cell or its hybridoma Monoclonal antibody.In other cases, from single cell clone, such as encoding antibody or its tau binding fragment or partial DNA point The growth of the eukaryotic host cell of son transfection prepares monoclonal antibody.It can also will encode the antibody and fragment of present disclosure Delivery of nucleic acids is used for cells expressing antibody and fragment by host subject into host subject.On October 15th, 1998 Described in the PCT Application No. WO98/44955 delivered for by the central nervous system of delivery of polynucleotides to host subject In and express the tactful example of anti-senilin antibody wherein.Any nucleic acid can be modified to increase internal stability.May Modification include but is not limited to 5' and/or 3' ends add flanking sequence；Thiophosphate or 2'O- methyl are used in skeleton Rather than phosphodiesterase key；And/or including nontraditional bases such as inosine, Q nucleosides (queuosine) and bosom fourth glycosides, and acetyl Base-methyl, it is thio-or other modified forms adenine, cytidine, guanine, thymidine and uridine.

On the one hand, antibody or tau binding fragment as described herein can compare physiological tau displayings to pathologic tau Higher affinity.

On the other hand, antibody or tau binding fragment as described herein can suppress tau-tau aggregations.

On the other hand, antibody or tau binding fragment as described herein, which can mediate, is absorbed and dropped by microglia Solve pathologic Protein tau.

On the one hand, antibody or tau binding fragment as described herein can compare physiological tau displayings to pathologic tau Higher affinity simultaneously suppresses tau-tau aggregations.

On the one hand, antibody or tau binding fragment as described herein can compare physiological tau displayings to pathologic tau Higher affinity, suppress tau-tau aggregations, and mediate and absorbed by microglia and degraded pathologic Protein tau.

Table 7 below, 8 and 9 outline CDR and the nucleotide sequence of variable region and the complete chain of some antibody described herein.

Table 7

Table 8

Table 9

In different groups of embodiment, the present invention provides the nucleic acid for encoding antibody as described herein or part thereof. Use GeneScript ' s proprietary technology codon optimizations encoding heavy chain variable region DC8E8VH, RHA, RHB, RHC, RHD, RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL and RHM and light chain variable district DC8E8 VK, RKA and RKB nucleic acid.At this In the context of application, codon optimization is the process of modified nucleotide sequence in the following manner, and the mode improves it true Expression, G/C contents in nucleus, RNA secondary structures, and translation, the amino acid sequence of its coding is not changed but.

In one embodiment, the present invention provides the nucleic acid for encoding humanised antibody heavy chain variable region as described herein (DNA or RNA).In one embodiment, nucleic acid include encoding heavy chain variable region RHA, RHB, RHC, RHD, RHE, RHF, RHG, Any one DNA of RHH, RHI, RHJ, RHK, RHL and RHM.These nucleic acid are presented in the following table.Displaying at least 80%, extremely Few 85%, at least 90%, at least 95%, and at least nucleic acid of 98% percentage identity is also in the range of the embodiment.

Table 10 below outlines a plurality of core of a plurality of full length sequence of the light chain corresponding to humanized antibody as described herein Acid sequence.

The constant K of complete humanization variable light	SEQ ID NO.
		RKA	141
RKB	142
		cDC8E8κ	143

Table 10

In another embodiment, the present invention provides the nucleic acid for encoding humanized antibody light chain variable region as described herein (DNA or RNA).In one embodiment, nucleic acid includes coding light chain variable region RKA and RKB any one DNA. Above present these nucleic acid.Displaying at least 80%, at least 85%, at least 90%, at least 95%, and at least 98% percentage The nucleic acid of homogeneity is also in the range of the embodiment.

Those skilled in the art will appreciate that, there are many codings such as this paper institutes in the result as degenerate The nucleotide sequence of the antibody stated or its tau binding fragment.In these nucleic acid some carry with it is as described herein any " wild The minimum homology of the nucleotide sequence of type " antibody or its tau binding fragment.Nevertheless, the difference in being selected due to codon Different and different nucleic acid is by specific consideration of the invention.

In one embodiment, the present invention provides the nucleic acid for encoding humanised antibody heavy chain variable region as described herein (DNA or RNA).In one embodiment, nucleic acid include encoding heavy chain variable region RHA, RHB, RHC, RHD, RHE, RHF, RHG, Any one DNA of RHH, RHI, RHJ, RHK, RHL and RHM, it connects human IgG1's constant region.Displaying at least 80%, at least 85%th, at least 90%, at least 95%, and at least nucleic acid of 98% percentage identity is also in the range of the embodiment.

In one embodiment, the present invention provides the nucleic acid for encoding humanised antibody heavy chain variable region as described herein (DNA or RNA).In one embodiment, nucleic acid include encoding heavy chain variable region RHA, RHB, RHC, RHD, RHE, RHF, RHG, Any one DNA of RHH, RHI, RHJ, RHK, RHL and RHM, it connects the constant region of human IgG 4.Displaying at least 80%, at least 85%th, at least 90%, at least 95%, and at least nucleic acid of 98% percentage identity is also in the range of the embodiment.

In another embodiment, the present invention provides the nucleic acid for encoding humanized antibody light chain variable region as described herein (DNA or RNA).In one embodiment, nucleic acid includes coding light chain variable region RKA and RKB any one DNA, its Connect people κ areas.Displaying at least 80%, at least 85%, at least 90%, at least 95%, and at least core of 98% percentage identity Acid is also in the range of the embodiment.

In another embodiment, the present invention provides the nucleic acid that each DC8E8 ' s CDR are encoded in a manner of codon optimization (DNA or RNA).

With showing that the percentage of at least 80%, such as 85%, 90%, 95% and 98% is same after preferred sequence optimal comparison Property nucleotide sequence represent that nucleotide sequence shows some modifications for reference nucleic acid sequence, e.g., particularly, lack, truncate, prolong Stretch, chimeric fusion and/or substitution, especially accurately.In some embodiments, these are following sequences, and it encodes and refers to sequence Row identical amino acid sequence, this is related to the degeneracy of genetic code, or complementary series, and it may be with reference sequences for example Under high stringency, particularly specific hybrid under the conditions of those defined below.

Hybridization represents to allow to maintain this side hybridized between two complementary DNA fragments with them under high stringency Formula selects the condition related to temperature and ionic strength.It is described above to define polynucleotides on the basis of purely illustrative The high stringency of the hybridization step of the purpose of fragment be it is favourable, it is as follows.

DNA-DNA or DNA-RNA hybridization is carried out with two steps：(1) containing 5X SSC, (1X SSC correspond to 0.15M The solution of NaCl+0.015M sodium citrates), 50% formamide, 7% lauryl sodium sulfate (SDS), 10X Denhardt ' s, In the phosphate buffer of 5% dextran sulfate and 1% salmon sperm DNA (20mM, pH 7.5) at 42 DEG C prehybridization 3 hours；(2) According to the length of probe, Preliminary Hybridization 20 hours is (i.e. at a certain temperature：For length>The probe of 100 nucleotides, 42 DEG C), then cleaned 2 times 20 minutes at 20 DEG C in 2X SSC+2%SDS, in 0.1X SSC+0.1%SDS at 20 DEG C Cleaning 1 time 20 minutes.For length>The probe of 100 nucleotides, carried out most in 0.1X SSC+0.1%SDS at 60 DEG C Cleaning afterwards 30 minutes.(Molecular cloning can be waited according to Sambrook:a laboratory manual,Cold Spring Harbor Laboratory；3rd edition, 2001) method described in, it is that above-mentioned determination is big by those skilled in the art Small polynucleotides, longer or shorter oligonucleotides adjustment high stringency conditions.

Expression system

Can with it is synthetically prepared or by any number of expression system known to persons of ordinary skill in the art prepare antibody and Its tau binding fragment.For this purpose, there is also provided following embodiments, it covers cloning vector, expression vector, Su Zhuxi Born of the same parents, and be transfected, convert or other heredity or recombinant modifieds are with the transgenic animals containing one or more above-mentioned nucleotide sequences (except people).In one embodiment, host cell is eucaryon.In another embodiment, host cell is protokaryon. In another embodiment, host cell expression one or more antibody and its tau binding fragments.Selected cell can be cultivated simultaneously And if desired, the protein product of target gene is separated from culture using routine techniques.In some embodiments, express System has been adapted to express antibody or its tau binding fragment with optimum level.In some embodiments, by based on contract Antibody expression company (for example, Lonza) expression antibody and its tau binding fragments, the company expressed using proprietary technology Antibody and its tau binding fragments are as service.In another embodiment, antibody and its tau knots are expressed in cell free system Close fragment.

The example of conventional use of antibody expression systems includes recombinant baculovirus, slow virus, protozoan (for example, true Core parasite Leishmania (Leishmania tarentolae)), Microbial Expression Systems, including based on yeast (such as finish Red yeast (Pichia pastoris), saccharomyces cerevisiae (Saccharomyces cerevisiae), yeast (lipolytica), Multiple-shaped nuohan inferior yeast (Hansenula polymorpha), aspergillus (Aspergillus) and trichoderma fungi) and based on bacterium (example Such as, Escherichia coli (E.coli), liquefaction pseudomonas fluorescens (Pseudomonas fluorescens), lactic acid bacteria (Lactobacillus), galactococcus (Lactococcus), Bacillus megatherium (Bacillus megaterium), withered grass bar Bacterium (Bacillus subtilis), Brevibacillus (Brevibacillus), corynebacterium glutamicum (Corynebacterium glutamicum)), Chinese hamster ovary (CHO) cell, CHOK1SVNSO (Lonza), BHK (children Logical sequence mouse kidney), PerC.6or Per.C6 (for example, Percivia, Crucell), HEK 293 not homology, Expi293F^TMCell (Life Technologies)、GenScript's YeastHIGH^TMTechnology (GenScript), people's neural precursor are thin Born of the same parents system AGE1.HN (Probiogen), plant (for example, corn, clover and tobacco), insect cell, bird egg, algae, and transgenosis The expression system of animal (for example, mouse, goat, sheep, pig, ox).

The glycosylation pattern of the antibody of these different systems expression is dramatically different according to expression system.For example, Chinese hamster ovary celI Glycosylate machine and people's glycosylation machine is more or less similar, also there are some different.In addition to the selection of host cell, in antibody Restructuring produce during influence glycosylated factor include growth pattern, culture medium preparation, culture density, oxygenation, pH, Purification schemes etc..It is proposed that a variety of methods change the glycosylation pattern realized in specific host biology, be included in oligosaccharides generation In the introducing that is related to or be overexpressed some enzyme (U.S. Patent numbers 5,047,335；5,510,261 and 5,278,299).Such as using Endoglycosidase (Endo H) removes glycosylation or certain form of glycosylation from glycoprotein enzymatic.Furthermore, it is possible to genetic modification Recombinant host cell is to be defect in certain form of polysaccharide is processed.These and similar techniques to be known in the art and It can be used for changing antibody and its tau binding fragment as described herein.

Pros and Cons of these different systems and known to those skilled in the art have been reviewed in the literature. Through describing some in these in the following documents, its is all incorporated herein with it entirely through reference：Chadd etc. Therapeutic antibody expression technology.Curr Opin Biotechnol.2001 Apr；12 (2):188-94；The Human antibody expression in transgenic such as Ma rats:comparison of chimeric IgH loci with human VH,D and JH but bearing different rat C-gene regions.J Immunol Methods.2013 Dec 31；400-401:78-86；The Monoclonal such as Zhang antibody expression in mammalian cells.Methods Mol Biol.2012；907:341-58.

Pharmaceutical composition and preparation

It provided herein is comprising humanized antibody as described herein or its tau binding fragment, and another component, such as carry The composition of body.What is be also provided herein is to include humanized antibody as described herein or its tau binding fragment, and excipient And/or the pharmaceutical composition of pharmaceutical carrier/treatment preparation.In one embodiment, carrier is not naturally occurring compound. In one embodiment, excipient is not naturally occurring compound.In another embodiment, diluent is not naturally to deposit Compound.In another embodiment, the preparation comprising humanized antibody as described herein or its tau binding fragment is not Containing naturally occurring compound, optionally in addition to water.

In one embodiment, by by the antibody with desired purity level or its tau binding fragment and optionally Pharmaceutically acceptable carrier, diluent, excipient or stabilizer mixing prepare controlling for antibody used according to the invention Preparation (it is the form of lyophilized formulation or the aqueous solution) is treated to be used to store and/or using (Remington's Pharmaceutical Sciences the 16th edition, Osol, A. are edited (1980)).In one embodiment, acceptable load Body, excipient or stabilizer are nontoxic to subject on used dosage and concentration, and including buffer solution such as phosphoric acid Salt, citrate and other organic acids；Antioxidant includes ascorbic acid and methionine；Preservative (such as hexadecyldimethylamine Base benzyl ammonium chloride；Chloor-hexaviet；Benzalkonium chloride, benzethonium chloride；Phenol, butanol or phenmethylol；Alkyl is to oxybenzene Formic acid esters such as methyl or propyl p-hydroxybenzoate；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols and m- cresols)；Low molecule Measure (less than about 10 residues) polypeptide；Protein, such as seralbumin, gelatin or immunoglobulin；For example poly- second of hydrophilic polymer Alkene pyrrolones；Amino acid such as glycine, glutamine, asparagine, histidine, arginine or lysine；Monose, disaccharides, and Other carbohydrate, including glucose, mannose, or dextrin；Chelating agent such as EDTA；Sugar as sucrose, mannitol, trehalose or Sorbierite；The ion balance of forming salt such as sodium；Metal composite (such as Zn- protein metal composite)；And/or nonionic Surfactant such as TWEEN, PLURONICS or polyethylene glycol (PEG).The example of the antibody preparation of lyophilized is described in WO In 97/04801, by reference to being clearly incorporated herein.

In other embodiments, preparation also includes surfactant.In other embodiments, preparation also includes surface Activating agent.The surfactant can for example be selected from detergent, ethoxylated castor oil, polyglycosylated (polyglycolyzed) glyceride, acetylated monoglyceride, sorbitan fatty acid ester, polyoxypropylene polyoxyethylene Blocked polymer (for example, poloxamer such as Pluronic.RTM.F68, poloxamer 188 and 407, Triton X-100), polyoxy Ethene sorbitan fatty acid ester, polyoxyethylene and polythene derivative such as alkylation and alkoxy derivative (tweens, Such as Tween-20, Tween-40, Tween-80 and Brij-35), monoglyceride or its ethoxylated derivative, diglyceride Or its polyoxyethylene deriv, alcohol, glycerine, agglutinin and phosphatide (such as phosphatidylserine, phosphatidyl choline, phosphatidyl second Hydramine, phosphatidylinositols, cardiolipin and sphingomyelins), the derivative (such as DPPA) and haemolysis of phosphatide The derivative of phosphatide is (for example, palmityl lysophosphatide-Serine and monoethanolamine, choline, the 1- acyls of serine or threonine Base-sn- glycerol-3-phosphates ester) and alkyl, alkoxy (Arrcostab), alkoxy (Arrcostab)-lysophosphatidylcholine and phosphatidyl The derivative of choline, such as the lauroyl of lysophosphatidylcholine, DPPC and myristoyl derivative, and pole Property head group modification, it is choline, monoethanolamine, phosphatidic acid, serine, threonine, glycerine, inositol, and positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatide serine and lysophosphatide threonine, and glycerophosphatide (such as cephalin), Glyceroglycolipid (such as galactopyranose glycosides), glycosyl sphingolipid (such as ceramide, gangliosides), dodecylphosphoric acid courage Alkali, egg lysolecithin, fusidic acid derivatives -- (such as ox sulphur dihydro fucidin etc.), long chain fatty acids and its N α-acylation of C6-C12 salt (such as oleic acid and octanoic acid), acylcarnitines class and derivative, lysine, arginine or histidine Derivative or lysine or arginic side chain acylated derivatives including lysine, arginine or histidine and neutral or acidity Times of the N α-acylated derivatives including a neutral amino acid of any combination of dipeptides of amino acid and two Charged acids N α-acylated derivatives, DSS (docusate sodium, CAS registration numbers [577-11-7]), the docusa of the tripeptides of what combination, CAS are stepped on Mark [128-49-4]), docusate potassium, CAS registration numbers [7491-09-0]), SDS (lauryl sodium sulfate or lauryl sulfate Sodium), Sodium Caprylate, cholic acid or derivatives thereof, bile acid and its salt and glycine or taurine conjugate, ursodesoxycholic acid, cholic acid Sodium, NaTDC, natrium taurocholicum, NaGC, N- cetyls-N, N- dimethyl -3- ammonium -1- propane sulfonic acid salt, Anion (alkyl-aryl-group-Sulfonates) monovalence surfactant, zwitterionic surfactant (such as N- alkyl-N, N- bis- Methyl ammonium -1- propane sulfonic acid salt, 3- chloro-acid amide base -1- propyl-dimethyl ammonium -1- propane sulfonic acid salt, cationic surfactant (quaternary ammonium base) (such as cetab, cetylpyridinium chloride), nonionic surface active agent (such as 12 Alkyl β-D- glucopyranosides), poloxamer (poloxamine) (such as Tetronic's), its be derived from successively by ring Ethylene Oxide and ethyleneoxide addition are to the tetrafunctional block copolymer of ethylenediamine, or the surfactant may be selected from imidazoline and spread out Biology or its mixture.In one embodiment, surfactant is not naturally occurring compound.These specific surfaces Each of activating agent constitutes the optional embodiment of present disclosure.

One embodiment provides antibody and/or the stabilization formulations of its tau binding fragment, and it is preferably comprised with salt or institute Select the phosphate buffer of salt, and anti-corrosion buffer solution and the preparation containing preservative, and suitable pharmacy or veterinary purpose Multipurpose preservative, antibody and/or its tau binding fragment in its pharmaceutically acceptable preparation.In an embodiment In, preservative contains preservative known at least one, or is optionally selected from least one phenol, m- cresols, p- cresols, o- Cresols, chloreresol, phenmethylol, phenylmercuric nitrate, Phenoxyethanol, formaldehyde, anesin, magnesium chloride (for example, hexahydrate), Alkyl nipagin esters (methyl, ethyl, propyl group, butyl etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and Thimerosal, or its mixture in diluent water.Any suitable concentration known in the art or mixture can be used, such as 0.001-5%, or any scope therein or value, e.g., but be not limited to 0.001,0.003,0.005,0.009,0.01,0.02, 0.03、0.05、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、 1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、 3.5th, 3.6,3.7,3.8,3.9,4.0,4.3,4.5,4.6,4.7,4.8,4.9, or any scope therein or value.It is non-limiting Example includes, preservative free, 0.1-2%m- cresols (for example, 0.2,0.3,0.4,0.5,0.9,1.0%), 0.1-3% phenmethylols (for example, 0.5,0.9,1.1,1.5,1.9,2.0,2.5%), 0.001-0.5% thimerosals (for example, 0.005,0.01), 0.001-2.0% phenol (for example, 0.05,0.25,0.28,0.5,0.9,1.0%), 0.0005-1.0% alkyl nipagin esters (for example, 0.00075,0.0009,0.001,0.002,0.005,0.0075,0.009,0.01,0.02,0.05,0.075, 0.09th, 0.1,0.2,0.3,0.5,0.75,0.9,1.0%) etc..In one embodiment, preservative or Determination of Preservatives be not It is naturally occurring compound.

In one embodiment, the antibody of present disclosure and its tau binding fragments, which can be incorporated into, is adapted to tested The pharmaceutical composition that person applies.In a conventional implementation, described pharmaceutical composition includes the antibody or its tau of the present invention Binding fragment and pharmaceutically acceptable carrier.In one embodiment, " pharmaceutically acceptable carrier " include it is any and All solvent, decentralized medium, coating, antibacterial agent and antifungal agent, isotonic agent and absorption delaying agent etc., it is physiologically Compatible.The additional examples of pharmaceutically acceptable carrier include water, salt solution, phosphate buffered saline solution, glucose, glycerine, second The one or more of alcohol etc., with and combinations thereof.In many cases, isotonic agent will be preferably included in the composition, such as sucrose, Polyalcohol such as mannitol, sorbierite or sodium chloride.Pharmaceutically acceptable carrier can further include less amount of auxiliary Material such as wetting agent or emulsifying agent, preservative or buffer, it improves the Storage period of antibody or its tau binding fragment or effective Property.

In one embodiment, the pharmaceutically acceptable salt of appropriate amount is used in preparation to cause preparation isotonic.Carry The example of body includes salt solution, Ringer's solution and glucose solution.In one embodiment, the pH of solution can be about 5- About 8.In another embodiment, pH is about 7- about 7.5.Other carriers include continuing containing antibody or its tau binding fragment Delivery formulations, such as the semipermeable matrices of solid hydrophobic polymers, the matrix is in molded article such as film, liposome or particulate Form.The matrix of sustained release as used herein is by following substances, can usually be hydrolyzed by enzymatic or acid/base or logical Cross matrix made of decomposition degradable polymer.Once being inserted into vivo, matrix is worked by enzyme and body fluid.It is expected that by The material of bio-compatible, such as liposome, polylactide (more lactic acid), polyglycolide (polymer of glycolic), more The common glycolide (copolymer of lactic acid and glycolic) of polylactides, polyanhydride, poly- (just) ester, polypeptide, hyaluronidase, collagen, sulphur Aching and limp ossein, carboxylic acid, aliphatic acid, phosphatide, polysaccharide, nucleic acid, amino acids, amino acid such as phenylalanine, tyrosine, different bright ammonia Acid, polynucleotides, polyethylene propylene, polyvinyl pyrrolidone and silicone select the matrix of sustained release.Preferable biodegradable Matrix be polylactide, polyglycolide or polylactide altogether glycolide (copolymer of lactic acid and glycolic) it One matrix.

It will be apparent for a person skilled in the art that some carriers can more preferably dependent on such as route of administration and The concentration for the antibody applied.

The composition of the present invention can be diversified forms.These are included for example, liquid, semisolid and solid dosage forms, such as liquid Liquid solution (for example, injectable and can be transfused the solution of (infusible)), dispersant or suspending agent, tablet, pill, pulvis, Liposome and suppository.In some embodiments, said composition can also include buffer solution (for example, neutral buffered saline or phosphorus Acid buffering salting liquid), carbohydrate (for example, glucose, mannose, sucrose or glucan), mannitol, protein, polypeptide Or amino acid such as glycine, antioxidant, chelating agent such as EDTA or Agifutol, adjuvant (for example, aluminium hydroxide) and/or anti-corrosion Agent.Or composition of the invention can be formulated as it is freeze-dried.Widely-known technique can also be used by antibody and its tau Binding fragment is encapsulated in liposome.

It is suitable for antibody or its tau combinations that internal applied dose form typically contains about 0.1 milligram-about 500 milligrams Fragment (active component)/unit or container.In these pharmaceutical compositions, active component is based on the gross weight of composition with weight Meter is generally by the amount presence with about 0.5-99.999%.

Therapeutic combination/preparation generally has to be sterile and stable under conditions of manufacture and storage.Composition can match somebody with somebody Solution, microemulsion, dispersant, liposome are made as, or is suitable for other ordered structures of high drug concentration.Sterile injectable is molten Liquid can be a kind of listed above by the way that reactive compound (i.e. antibody or its tau binding fragment) is had with the amount incorporation of needs Prepared in composition or the suitable solvent of its combination (if desired, being then filtration sterilization).Generally, dispersant is by by activity Prepared in compound incorporation sterile carrier, the sterile medium contain basic dispersion medium and it is listed above those In needs other compositions.In the case of the sterile powder for preparing sterile injectable solution, preferable preparation method It is to be dried in vacuo and freeze dried, it produces active component from its solution being previously sterile filtered and adds any extra desired composition Pulvis.For example, can by using coating such as lecithin, by the granular size required for maintaining in the case of dispersant, And appropriate solution mobility is maintained by using surfactant.The examination that can be absorbed by including delay in the composition Agent, such as Monostearate and gelatin cause the delay of Injectable composition to absorb.

Preferable dosage form depends on expected mode of administration and treatment use.It is familiar to the person skilled in the art to be used to survey The program of the fixed dosage.Typical composition is the form of injectable or the solution that can be transfused, such as with utilizing other antibody quilts Those similar compositions of dynamic immune people.Most typical method of application be parenteral (for example, intravenous, subcutaneous, intraperitoneal, It is intramuscular).In preferred embodiments, antibody is applied by intravenous infusion or injection.In another preferred embodiment In, antibody is applied by intramuscular or hypodermic injection.

The antibody and its tau binding fragments of the present invention can be applied by a variety of methods known in the art, although right In many treatment uses, preferable route of administration/mode is intravenous injection or infusion.It will be appreciated by the skilled person that apply Approach and/or mode will change according to desired result.In certain embodiments, antibody or its tau binding fragment can be with Being prepared with carrier, the carrier will protect compound from quick release, such as the preparation of controlled release, including implant, thoroughly Skin patch and the delivery system of microencapsulation.Biodegradable, bio-compatible polymer, such as ethylene-vinyl acetate can be used Ester, polyanhydride, polyglycolic acid, collagen, poe and PLA.The method that many is used for the preparation of such preparation is to obtain patent Or it is generally known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson, editor, Marcel Dekker, Inc., New York, 1978.

In certain embodiments, antibody of the invention or its tau binding fragment can orally administer, for example, together with lazy Property diluent or assimilable edible carrier.Antibody or its tau binding fragment (if desired, and other compositions) can also be encapsulated In duricrust or soft shell gelatin capsules, it is compressed into tablet, or is directly incorporated into the diet of subject.Applied for oral medication With antibody or its tau binding fragment can be mixed with excipient and with absorbable tablet, buccal tablet (buccal tablet), ingots Agent, capsule, elixir, suspending agent, syrup, the form of wafer (wafer) use.In order to by way of except parenteral administration Using the antibody or its tau binding fragment of the present invention, it may be necessary to be coated with the antibody or its tau with the material that it is inactivated is prevented Binding fragment applies the antibody or its tau binding fragment together.

Certain embodiments of the present invention provide antibody or its tau binding fragment with through blood-brain barrier.Some nerves become Property disease, including AD and correlation tau lesions it is related to the permeability increase of blood-brain barrier so that the antibody or its tau knot Fragment is closed easily to be imported in brain.When blood-brain barrier keep it is complete when, exist method known to some fields be used for will Molecular transport passes through the blood-brain barrier, including but not limited to physical method, the method based on fat and based on acceptor and passage Method.

The physical method of transhipment antibody or its tau binding fragment through blood-brain barrier includes, but are not limited to entirely around blood Brain barrier, or be open by being created in blood-brain barrier.Include but is not limited to be injected directly into brain (referring to example around method Such as, Papanastassiou etc., Gene Therapy 9:398-406 (2002)) and delivery apparatus is implanted into (referring to example in brain Such as, Gill etc., Nature Med.9:589-595(2003)；With Gliadel Wafers.TM., Guildford Pharmaceutical).The method that opening is created in barrier includes, but are not limited to ultrasound (see, for example, US publication 2002/0038086), osmotic pressure is (for example, by applying Hypertonic mannitol solution (Neuwelt, E.A., Implication of the Blood-Brain Barrier and its Manipulation, the 1＆2 volumes, Plenum Press, N.Y. (1989))), lead to Cross such as bradykinin or permeabilization agent A-7 permeabilizations (see, for example, U.S. Patent number 5,112,596,5,268,164,5,506, 206 and 5,686,416), and the carrier transfected neurons using the gene containing encoding antibody or its tau binding fragment, it is horizontal Across blood-brain barrier (see, for example, U.S. Patent Publication No. 2003/0083299).

Transhipment antibody or its tau binding fragment based on fat include, but are not limited to be coupled across the method for blood-brain barrier Encapsulated antibody or its tau binding fragment, the blood vessel endothelium of the liposome combination blood-brain barrier in the liposome of its active fragment On acceptor (see, for example, U.S. Patent Application Publication No. 20020025313), and (referring to example in low-density lipoprotein particle Such as, U.S. Patent Application Publication No. 20040204354) or apo E in (see, for example, U.S. Patent Application Publication No. 20040131692) coated antibody or its tau binding fragment.

Transhipment antibody or its tau binding fragment based on acceptor and passage include across the method for blood-brain barrier, but unlimited In using glucocorticoid blocking agent to increase the permeability of blood-brain barrier (see, for example, U.S. Patent Application Publication No. 2002/ 0065259th, 2003/0162695 and 2005/0124533)；Potassium-channel is activated (see, for example, U.S. Patent Application Publication Number 2003/0073713)；With transferrins coated antibody and the activity for adjusting one or more TfRs (referring to example Such as, U.S. Patent Application Publication No. 2003/0129186), and cationized antibodies (see, for example, U.S. Patent number 5,004, 697)。

Various other methods apply compound into brain to realize as is generally known in the art.For example, antibody or its tau bonding pad Section can be applied will be down to most to the influence of brain parenchym by direct ventricle be interior or intrathecal injection preferably through slow infusion It is small.Desired antibody or its tau binding fragment can also use the slow release implant in brain, or produce antibody or its tau The implantation recombinant cell delivering of binding fragment.Blood-brain barrier (BBB) can carry out permeabilization and be applied with antibody or its tau binding fragment With to allow antibody or its tau binding fragment to move through BBB.Permeabilization agent includes bleeding agent, such as Hypertonic mannitol solution, or another Permeabilization agent such as bradykinin (bradykinin), alkyl glycerol, ultrasound, electromagnetic radiation or parasympathetic innervation.

In addition, and do not fettered by any specific mechanism, also it had been thought that likely the antibody in blood is from brain Removing has " pond sample " effect in its target protein.See, for example, the U.S. Published Application U.S. 20110158986, [0017] section. If it is the case, antibody and its tau binding fragments are possibly used for removing pathologic tau from brain into circulating, effectively Prevent it and further damage is caused to neuronal cell and tissue.

Disclosed supplement or combined activity compound or therapeutic agent can also be incorporated into composition elsewhere in the application In, be administered simultaneously, or with antibody or its tau binding fragment continuous administration.In certain embodiments, antibody of the invention or its Tau binding fragments and one or more extra therapeutic agent co-formulations and/or apply together.These extra therapeutic agents Can also chemically conjugated wherein described antibody or its tau binding fragment.

In one embodiment, there is provided there are formula (A)-(L)-(C) immunoconjugates, wherein：(A) it is claim The antibody of 1-51 any one or its binding fragment；(L) it is joint；And (C) is reagent；And wherein described joint (L) is even Meet (A) and (C).In one embodiment, (C) is effector molecule, such as therapeutic agent, preparation, detectable agent, or diagnosis Agent.In some embodiments, these conjugates are referred to herein as antibody-drug-conjugate (ADC).

As used herein, (L) joint is to be used to connect (A) to the molecule of (C).The joint can be with antibody and effect point Son forms covalent bond.Suitable joint be well known to those skilled in the art and including but not limited to straight or branched carbon joint, Heterocycle carbon joint or peptide linker.When antibody and effector molecule are polypeptides, joint can be by its side base (for example, by with half The disulfide bond of cystine) connect and compose amino acid.However, in preferred embodiments, joint can connect end amino acid α carbon amino and carboxyl.

In some cases, when conjugate has arrived at its target site, it is expected to dissociate effector molecule from antibody.Thus, In such cases, immunoconjugates will include the connection that can be cut near target site.Enzymatic activity or target can be passed through Nearby the condition residing for immunoconjugates promotes the cutting of joint with from antibody release effects molecule to intracellular or target site.Still In other right embodiments, connector unit is not cleavable and for example discharges medicine by antibody degradation.

Many differential responses can be obtained to be used to medicine and/or joint being attached to antibody or its fragment.This frequently by The amino acid residue of antibody molecule, including the amido of lysine, the free carboxy of glutamic acid and aspartic acid, the mercapto of cysteine The reaction of some of base and aromatic amino acid is completed.One of the most frequently used non-specific method of covalent attachment is to connect Connect the Carbodiimide reaction of the carboxyl (or amino) of compound and the amino (or carboxyl) of antibody.In addition, difunctional dose such as two Aldehyde or imino-ester have been used to connect the amino of compound and the amino of antibody molecule.It can be also used for medicine being attached to anti- On body is schiff base reaction.This method includes the periodate oxidation of the medicine containing ethylene glycol or hydroxyl, therefore is formed right The aldehyde reacted afterwards with bonding agent.The formation for being attached by the amino of schiff bases and bonding agent occurs.Isothiocyanate can also be used Make coupling agent to be used to medicine being covalently attached to bonding agent.

In some embodiments, joint can by intracellular environment (such as lysosome or inclusion body or cell membrane it is small recessed (caveolea) in) present in cutting agent cutting.Joint can be such as peptidyl linkers, and it can be by intracellular peptase or albumen Enzyme, including but not limited to lysosome or inclusion body protease are cut.In some embodiments, peptidyl linkers are at least two ammonia Base acid length or at least three amino acid longs.Cutting agent can include cathepsin B and D and fibrinolysin, it is known that its is complete Portion hydrolyzes dipeptide medicament derivative, cause the active medicine in target cell discharge (see, for example, Dubowchik and Walker, 1999,Pharm.Therapeutics 83:67-123).Most typically peptidyl linkers, it can be expressed 191P4D12's Cleavage present in cell.Other examples of such joint are described in such as U.S. Patent number 6,214,345.Specific real Apply in scheme, can be Val-Cit joints or Phe-Lys joints by the peptidyl linkers of intracellular protein cleavage (see, for example, the U.S. The patent No. 6,214,345, which depict the synthesis of adriamycin and Val-Cit joints).Discharged and treated using intracellular protein enzyme hydrolysis One advantage of agent is that the reagent generally serum stability of decay and conjugate is typically high when being conjugated.

In other embodiments, cleavable joint is pH sensitivities, i.e., sensitive to the hydrolysis under some pH value.It is logical Often, joint sensitive pH is hydrolyzable in acid condition.It is, for example, possible to use what can be hydrolyzed in lysosome is sour unstable Joint (for example, hydrazone, semicarbazones, thiosemicarbazone, cis-rhizome of Chinese monkshood acid amides, ortho esters, acetal, ketal etc.).(referring to For example, U.S. Patent number 5,122,368；5,824,805；5,622,929；Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123；Neville etc., 1989, Biol.Chem.264:14653-14661.) such connect Head is relatively stable under conditions of neutral ph, as in blood, but less than (the approximations of lysosome of pH 5.5 or 5.0 It is unstable when pH).In certain embodiments, hydrolyzable joint is thioether linker (e.g., for example, being attached to by acylhydrazone key Thioether on therapeutic agent (see, for example, U.S. Patent number 5,622,929).

In still other embodiments, joint is cleavable (for example, disulfide bond joint) under the reducing conditions.It is a variety of Disulfide bond joint is known in the art, including for example can use SATA (N- succinimido-S- thioacetic acids acetonyl ester), SPDP (N- succinimidos -3- (2- pyridines two are thio) propionic ester), the SPDB (N- succinimidos -3- (sulphur of 2- pyridines two Generation) butyrate) and SMPT (N- succinimido-oxygen carbonyls-Alpha-Methyl-α-(2- pyridine radicals-two is thio) toluene)-, Those of SPDB and SMPT formation.(see, e.g., Thorpe etc., 1987, Cancer Res.47:5924-5931； Wawrzynczak etc., In Immunoconjugates:Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W.Vogel is edited, Oxford U.Press, and 1987. referring further to U.S. Patent number 4,880, 935)。

In still other specific embodiments, joint be malonate joint (Johnson etc., 1995, Anticancer Res.15:1387-93), maleimidobenzoyl joints (Lau etc., 1995, Bioorg-Med-Chem.3 (10):1299- , or 3'-N- amide analogues (Lau etc., 1995, Bioorg-Med-Chem.3 (10) 1304):1305-12).

In still other embodiments, connector unit is not cleavable and discharges medicine by antibody degradation.(ginseng See US publication 2005/0238649).

In one embodiment, joint is substantially insensitive to extracellular environment.As used herein, under the background of joint It is " substantially insensitive to extracellular environment " to represent when antibody-drug conjugate compound is present in extracellular environment (for example, blood plasma) When, no more than about 20%, typically no more than about 15%, more generally no more than about 10% in antibody-drug conjugate compound, and About 5%, no more than about 3% is even more typically no more than, or no more than about 1% joint is cut.Such as can by antibody- Drug conjugate compound incubates the time limit scheduled time (for example, 2,4,8,16 or 24 hours) with blood plasma, is then quantitatively deposited in blood plasma Free drug amount it is whether substantially insensitive to extracellular environment to determine joint.

In other non-mutually exclusive embodiments, joint promotes cell internalizing.In certain embodiments, when conjugated During therapeutic agent (in the environment of joint-therapeutic agent portion in antibody-drug conjugate compound as described herein), joint Promote cell internalizing.

In WO 2004-010957, US publication 2006/0074008, US publication 20050238649, and the U.S. This group is described in publication number 2006/0024317 (it is each expressly incorporated herein by reference with its entirety and for all purposes) The various exemplary joint that compound and method can use.

Some examples for the antibody-drug conjugates (ADC) that presently, there are in clinic are found in Feng, Y. etc. Conjugates of Small Molecule Drugs with Antibodies and Other Proteins.Biomedicines 2014,2,1-13；doi:10.3390/biomedicines2010001.

In view of it has been reported that a large amount of methods are used for a variety of therapeutic agents, preparation, detectable agent, diagnosticum, radiation Diagnostic compounds, radiotherapeutic compound, medicine, toxin and other reagents are attached in antibody and its fragment, art technology Personnel can determine suitable method and be used to indicated reagent being attached to antibody or its tau binding fragment.In another implementation In scheme, (A) and (C) is bonded directly to one another.

In another embodiment, (L) is spacer group or linking group, such as more aldehyde, glutaraldehyde, EDTA Or diethylene triamine pentacetic acid (DTPA) (DPTA) (EDTA).Other technologies for antibody or fragment to be connected to another compound include making With N- succinimidos -3- (2- pyridine radicals-thio) propionic ester (SPDP) and succinimido 4- (N- maleimide first Base) hexamethylene -1- carboxylates (SMCC) or derivative (if peptide lacks sulfydryl, this can by add cysteine residues come There is provided) form disulfide bond.These reagents produce disulfide bond in them and a protein peptide cysteine residues, and by relying ammonia Other free amine groups in ε amino or other amino acid on acid form amido link.Immun.Rev.62,185 (1982) is described A variety of such disulphide/acid amides forming agents.Other bifunctional coupling agents form thioether rather than disulfide bond.These many thioethers Forming agent is commercially available and including 6-maleimidocaproic acid, 2- bromoacetic acids, and 2- iodoacetic acid, 4-- The reaction ester of (N- maleimido-methyls) hexamethylene -1- carboxylic acids.Can be by by they and succinimide or 1- hydroxyls Base -2- nitro -4- sulfonic acid, sodium salt are combined to activate carboxyl.

Drugloading rate is represented by p and is the average (for example, A-L-Dp) of the drug moiety of each antibody in molecule.Carry medicine Amount can change between 1-20 drug moiety (D)/antibody.The ADC of the present invention includes sewing with 1-20 drug moiety scope The collection of antibodies of conjunction.It can be characterized by conventional meanses such as mass spectrum and ELISA measure every in the ADC preparations from conjugation reaction The average of the drug moiety of individual antibody.The ADC of p units distributed number can also be determined.In some cases, can pass through Means such as electrophoresis is completed to utilize separation, purifying and sign of other drugloading rates to homogeneity ADC, and wherein p is from a certain of ADC Value.

For some antibody-drug conjugates, p can be limited by the quantity of attachment site on antibody.For example, wherein When attachment is cysteine sulfydryl, as in exemplary above, antibody can have only one or several half Guang ammonia Sour sulfydryl, or the abundant reactive sulfydryl that be able to can adhere to only one or several joints.In certain embodiments, Higher drugloading rate, such as p>5 can cause the assembling, do not dissolve of some antibody-drug conjugates, toxicity, or cell permeable The forfeiture of property.In certain embodiments, ADC of the present invention drugloading rate is in about 1- about 8；About 2- about 6；About 3- about 5；About 3- about 4； About 3.1- about 3.9；About 3.2- about 3.8；About 3.2-3.7；About 3.2- about 3.6；About 3.3- about 3.8；Or about 3.3- about 3.7 scope It is interior.Really, have shown that the optimal proportion of the drug moiety of each antibody can be less than 8, and can be for some ADC About 2- about 5.Referring to U.S. Patent number 7,498,298.

In certain embodiments, the drug moiety conjugation of antibodies of theoretical maximum amount is less than during conjugation reaction.Antibody Such as lysine residue can be contained, it does not react with agent-linker intermediate or linker reagents, as discussed below.Usually, Antibody does not contain many free and reactive cysteine sulfydryl, and it connects drug moiety；Really most of half Guangs in antibody Propylhomoserin sulfhydryl residue exists with disulfide bond.In certain embodiments, reducing agent such as dithiothreitol (DTT) (DTT) or three can be utilized Carbonylethyl hydrogen phosphide (tricarbonylethylphosphine) (TCEP), reduced under partially or completely reducing condition anti- Body is to produce reactive cysteine sulfydryl.In certain embodiments, antibody is subjected to Denaturing with the reactive nucleophilic of exposure Group such as lysine or cysteine.

Such as pass through the following load (medicine/antibody ratios) for controlling ADC by different way：(i) limit in agent-linker Between thing or linker reagents relative to the molar excess of antibody, (ii) limitation conjugation reaction time or temperature, (iii) part or limitation Reducing condition is used for cysteine sulfydryl modification, (iv) by the amino acid sequence of recombinant technique engineered antibody, so as to modify half The quantity of cystine and position are used for the quantity and/or position (such as this paper and WO2006/034488 of control joint-medicine attachment Disclosed in the thioMab or thioFab that prepare).

It should be understood that more than one nucleophilic group and agent-linker intermediate or linker reagents then with drug moiety reagent During reaction, then products therefrom is the mixture of ADC compounds, and there are one or more drug moieties to be attached on antibody for it Distribution.Each resist can be calculated from mixture by binary ELISA TPPAs (it is special and special to medicine to antibody) The par of body medicine.Each ADC molecules and mutual by HPLC, such as hydrophobicity can be identified in the mixture by mass spectrum Action chromatography, which is separated, (see, for example, Hamblett, K J., waits " Effect of drug loading on the pharmacology,pharmacokinetics,and toxicity of an anti-CD30 antibody-drug conjugate,"Abstract No.624,American Association for Cancer Research,2004 Annual Meeting, Mar.27-31,2004, Proceedings of the AACR, volume 45, in March, 2004；Alley, S.C., wait " Controlling the location of drug attachment in antibody-drug conjugates,"Abstract No.627,American Association for Cancer Research,2004 Annual Meeting, 27-31 days in March, 2004,2004, Proceedings of the AACR, volume 45,2004 3 Month).In certain embodiments, the homogeneity with single load value can be separated from conjugation mixture by electrophoresis or chromatogram ADC。

In some example embodiments, antibody as described herein or its tau binding fragment can be with being also used for preventing And/or treatment AD one or more added compound co-formulations and/or co-administration.These include, but are not limited to be used for The compound of AD actively and passively immunization therapies, such as beta-amyloid peptide (for example, N- ends amyloid-beta peptide), tau Peptide, it may or may not be conjugated with other compounds, such as the diphtheria toxin of mutation；For the antibody of beta amyloid albumen, such as Bapineuzumab, solaneuzumab, gantenerumab, crenezumab, ponezumab, and IVIG immunoglobulins, Target other immunization therapies of A β oligomers, other tau antibody, prevent tau hyperphosphorylation compound, and targeting tau Other actively and passively immunization therapies of aggregation.It is used for combined therapy using antibody as described herein and tau binding fragments Other drugs are amyloid-beta peptide aggregation inhibitor (for example, Homotaurine (Tramiprosate)), inhibitors of gamma-secretase (for example, Si Maxite (semagacestat)), and gamma secretase modulators (tarenflurbil).In addition, the one of the present invention Kind or Multiple Antibodies can be used for combining with two or more above-mentioned therapeutic agents.In the early stage of disease, combined therapy can be with It is favourable.Combined therapy is also advantageous in the relatively late stage of disease, such as hAb and growth factor and other biological activity Molecule includes the combination of neuron plasticity and regeneration.Such combined therapy may be advantageously employed applying for lower dosage and treat Agent, thus avoid possible toxicity or the complication related to a variety of monotherapies.It is as described herein according to related embodiment Antibody or its tau binding fragment are used to combining that (one kind is administered alone, other it with selected from following at least one composite reagent Before, simultaneously or afterwards apply)：Acetylcholinesteraseinhibitors inhibitors (for example, donepezil, rivastigmine, galanthamine, Tacrine, nutritious supplementary pharmaceutical), N-methyl-D-aspartate (NMDA) receptor antagonist (for example, Memantine), DNA repair suppression Preparation (for example, pirenzepine or its metabolin), transition metal chelator, growth factor, hormone nonsteroidal and-inflammatory drug (NSAID), the suppression of inhibitor, protein kinase that antioxidant, lipid lowering agent, selective phosphodiesterase inhibitors, tau assemble Agent, the inhibitor of resist mitochondria abnormal drug, neurotrophic factor, the inhibitor of heat shock protein, lipoprotein combination phospholipase A₂、 And its any pharmaceutically acceptable salt.In one embodiment, using antibody and/or its tau binding fragment treatment with Using the therapeutic combination of anticholinesterase (ChEI) and/or Memantine, it provides appropriate symptom benefit.In an embodiment party In case, combined therapy agent is selected from inhibitor, 3- amino the third sulphurs of -1- that anti-apoptotic compound, metal-chelator, DNA are repaired Sour (3APS), the disulfonic acid of 1,3- third (1,3PDS), Secretase inhibitors, beta-secretase inhibitors, inhibitors of gamma-secretase, β-shallow lake Powdered protein peptide, amyloid beta antibody, neurotransmitter, beta sheet switch (breaker), anti-inflammatory molecular, and cholinesterase Inhibitor.In one embodiment, anticholinesterase is Tacrine, rivastigmine, donepezil, galanthamine, Or nutritious supplementary pharmaceutical.In another embodiment, extra therapeutic agent is selected from BACE inhibitor；Muscarinic antagonist；Cholinester Enzyme inhibitor；Inhibitors of gamma-secretase；Gamma secretase modulators；HMG-CoA reductase inhibitor；Non-steroidal antiinflammatory； N-methyl-D-aspartate receptor antagonist；Anti-amyloid antibodies；Vitamin E；Nicotinic acetylcholine receptors alpha7； CB1 receptor inverse agonists or CB1 receptor antagonists；Antibiotic；Growth hormone secretagogues；Histamine H 3 antagonists；AMPA excitements Agent；PDE4 inhibitor；GABA_AInverse agonist；The inhibitor of amyloid aggregation；Glycogen synthase kinase beta inhibitor；α points Secrete the accelerator of enzymatic activity；PDE-10 inhibitor and cholesterol absorption inhibitor.

It can be appropriately used for being described in WO with other compounds that antibody as described herein and tau binding fragments combine (referring particularly to page 16 and page 17) in 2004/058258, it include medicine target (the 36-39 pages), Alkanesulfonic acids and alkanolsulfuric acids (the 39-51 pages), anticholinesterase (51-56 Page), nmda receptor antagonist (the 56-58 pages), estrogen (the 58-59 pages), nonsteroid anti-inflammatory drugs anti-inflammatory agent (60-61 Page), antioxidant (the 61-62 pages), Pexoxisome proliferator activated acceptor (PPAR) antagonist (the 63-67 pages), Cholesterol-lowering agent (the 68-75 pages)；Amyloid inhibitors (the 75-77 pages), amyloid form inhibitor (77- Page 78), metal-chelator (the 78-79 pages), antipsychotic drug and antidepressants (the 80-82 pages), nutritious supplementary pharmaceutical (83- Page 89) and increase brain in bioactive substance availability compound (referring to the 89-93 pages) and prodrug the (the 93rd and 94 Page).

In one embodiment, antibody and/or its tau binding fragment are used for the current standard group with treatment in treatment Close, it includes anticholinesterase and Memantine (Namenda) NMDA activators.

The present invention pharmaceutical composition can include " therapeutically effective amount " or " prevention effective dose " antibody of the invention or Its tau binding fragment." therapeutically effective amount " refers on dosage and for the time cycle of needs, effectively realizes desired The amount for the treatment of results.The antibody of therapeutically effective amount or its tau binding fragment can change according to following factor, such as the disease of individual State, age, sex and body weight, and antibody or its tau binding fragment trigger the ability for wanting response in individual.Treatment has Effect amount or a kind of following amounts, wherein beneficial therapeutic effect exceed antibody or its tau binding fragment any detrimental effect or Deleterious effects." prevention effective dose " refers on dosage and for the time cycle of needs, effectively realizes desired prevention As a result amount.It is used for subject before disease or in the early stage of disease generally, due to preventive dose, therefore prevention has Effect amount can be less than therapeutically effective amount.

Dosage regimen can be adjusted to provide optimal expectation response (for example, therapeutic or preventative response).Example Such as, it can apply infusion scheme or quick filling, some broken doses can be applied with the time or can be according to treatment situation It is urgent indicated to proportionally reduce or incremental dose.In order to easily apply the uniformity with dosage, it may be particularly advantageous to dosage Unit form prepares parenteral composition.Dosage unit form as used herein, which refers to, to be suitable as subject to be treated Single dose material on the unit (physically discrete units) that separates；Per unit, which contains, to be computed producing The reactive compound and required pharmaceutical carrier of the scheduled volume of raw desired therapeutic effect.To dosage unit form of the invention Specific characteristic and to be achieved specific treatment or prevention effect of the specification by (a) reactive compound, and (b) are allocating such be used for Limitation intrinsic in the field of the reactive compound of sensitiveness is specified in treatment individual and directly depends on them.

Should pay attention to dose value can with the type and the order of severity of the patient's condition to be mitigated difference.Further it should be understood that pair In any particular subject, specific dosage regimen over time according to individual need and should apply composition or instruct composition The professional judgement of the people of administration and adjusted, and dosage range described herein is exemplary only and is not intended to limitation The scope of claimed composition or practice.

For example, the effective dose of the composition of present disclosure for treating situation described below is according to many factors Change, the factor include using means, target site, patient physiological status, no matter patient is people or animal, applied Other medicaments, and treatment are preventative or curative.Usually, patient is people.Therapeutic dose needs titrated next excellent Change security and efficiency.Therefore, generally turn into multidose in a period of time using the treatment of antibody or its tau binding fragment It is required.

For people antibody of the invention or its tau binding fragment treatment or prevention effective dose exemplary, non-limitative Scope is 0.1-20mg/kg, more preferably 1-10mg/kg.In a preferred embodiment, at least three months, preferably at least With the dosage administration of antibodies of multidose, and 1 and 10mg/kg in a period of six months.Optionally, with 0.01-0.6mg/kg's Dosage and weekly and mensal frequency administration of antibodies or its tau binding fragment.Optionally, with 0.05-0.5mg/kg Dosage administration of antibodies or its tau binding fragment.Optionally, tied with 0.05-0.25mg/kg dosage administration of antibodies or its tau Close fragment.Optionally, with weekly to 0.015-0.2mg/kg dosage administration of antibodies or its tau bonding pad once every two weeks Section.Optionally, with weekly to 0.05-0.15mg/kg dosage administration of antibodies or its tau binding fragment once every two weeks. Optionally, with weekly 0.05-0.07mg/kg dosage administration of antibodies or its tau binding fragment.Optionally, with every Mondays Secondary 0.06mg/kg dosage administration of antibodies or its tau binding fragment.Optionally, with the agent of 0.1-0.15mg/kg once every two weeks Measure administration of antibodies or its tau binding fragment.Optionally, with monthly 0.1-0.3mg/kg dosage administration of antibodies or its tau Binding fragment.Optionally, with monthly 0.2mg/kg dosage administration of antibodies or its tau binding fragment.Optionally, annual one Secondary administration of antibodies or its tau binding fragment.Optionally, applied with 1-40mg dosage and weekly and mensal frequency Antibody or its tau binding fragment.Optionally, with 5-25mg dosage administration of antibodies or its tau binding fragment.Optionally, with 2.5-15mg dosage administration of antibodies or its tau binding fragment.Optionally, with the weekly agent to 1-12mg once every two weeks Measure administration of antibodies or its tau binding fragment.Optionally, applied with the weekly dosage to 2.5-0.2mg/kg once every two weeks Antibody or its tau binding fragment.Optionally, with weekly 2.5-5mg dosage administration of antibodies or its tau binding fragment.Appoint Selection of land, with weekly 4-5mg dosage administration of antibodies or its tau binding fragment.Optionally, with 7-10mg's once every two weeks Dosage administration of antibodies or its tau binding fragment.

In other passive immunity embodiments using antibody as described herein or tau binding fragments, dosage is about 0.0001-100mg/kg, and more generally change in the scope of 0.01-5mg/kg places subject weight.In some applications, Can be with least dosage of 0.1mg/kg body weight, the dosage of at least 0.5mg/kg body weight, 1mg/kg body weight, or 0.1-10mg/kg Any combinations administration of antibodies of the dosage of body weight or the amount of its tau binding fragment.In certain methods, at least one moon, at least With multidose (equal or different) administration of antibodies or its tau binding fragment in a period of 3 months, or at least six moon.It is any to control Dose population during treatment can be such as 4 and 6, although other quantity can be used according to factor discussed above.It can pass through Any method monitoring treatment being discussed further below.

It is suitable to be determined by checking using the experience of other therapeutic monoclonal antibodies developed already Antibody preparation.For example, have shown that some monoclonal antibodies in clinical scenarios effectively, as Rituxan (Rituximab), Herceptin (trastuzumab), Xolair (omalizumab), Bexxar (tositumomab), Campath (alemtuzumab), (pa profit pearl is single by Zevalin, Oncolym, Remicade (infliximab), Lucentis (ranibizumab), Synagis It is anti-), Soliris (Yi Kuli monoclonal antibodies), Kadcyla (ado- trastuzumab emtansine), Avastin (bevacizumab), Erbitux (Cetuximab), Simponi (usury monoclonal antibody), Tysabri (natalizumab), Mabthera (Rituximab), Stelara (excellent spy gram monoclonal antibody), general bolster monoclonal antibody and Aducanumab, and the antibody of present disclosure can use similar system Agent.For example, Dan Ke can be supplied with the concentration of 10mg/mL in 100mg (10mL) or 500mg (50mL) disposable bottle Grand antibody, the monoclonal antibody is on 9.0mg/mL sodium chloride, 7.35mg/mL Trisodium citrate dihydrates, the poly- mountains of 0.7mg/mL IV administrations are formulated in pear ester 80 and sterile water for injection.PH is adjusted to 6.5.In another embodiment, comprising about 20mM sodium citrates, about 150mM NaCl, pH about 6.0 preparation in supply antibody.

The method for the treatment of and prevention

Antibody as described herein can be used for a variety for the treatment of and prevention methods of AD and related tau lesions to composition.Remove Outside the favorable property of the immunogenicity reduced, these antibody compare parent mouse DC8E8 antibody to disease tau to be had at least 80% binding affinity.Mouse DC8E8 is characterized extensively in WO2013/041962, wherein showing that it possesses mould in AD bodies Therapeutic properties in type.Therefore, have it is rational according to humanization DC8E8 is believed also by people AD treatment and prevention, together Shi Keneng induces less adverse immune response.

In one embodiment, methods described includes applying antibody as described above to subject/patient, encodes them Nucleic acid, or pharmaceutical composition.In prophylactic use, to be enough to eliminate or reduce disease risks, its seriousness is reduced, or prolong It starts late, including the biochemistry of disease, histology and/or behavior symptom, its complication in the presentation of disease generating process With the amounts of intermediate pathological phenotypes to easily have by Alzheimer's or another tau lesions or in addition Alzheimer's or The patient of another tau lesions risk applies pharmaceutical composition or medicament.In therapeutic application, to be enough to cure, or at least portion Divide and prevent disease symptomses (biochemistry, histology and/or behaviouristics), including its complication and centre disease in disease generating process The amount for managing phenotype applies composition or medicament to the patient for suspecting or suffering from already the disease.

As defined above, treatment includes, to disease, having disease symptomses or having aptitudinal subject for disease and answer With or using therapeutic agent, it is therefore an objective to cure, recover, mitigate, alleviate, change, remedy, improve, improve or influence disease, disease Symptom or the tendency for disease.In addition, the disease of the anti-tau antibody of humanization or its tau binding fragment composition is used with lacking Shape is compared, if present disclosure composition individually or combined with another therapeutic agent healing, recovery, mitigation, alleviation, change, At least one symptom of another tau lesions remedied, improve, improve or influenceed Alzheimer's or treat, then It is believed that result is effective treatment to potential illness, it is cured, recovers, mitigates, delays in spite of all condition symptoms Solve, change, remedy, improve, improve or influence.

" risky " individual possibility may not detectable disease, and before treatment method as described herein May or may be without the detectable disease of displaying." risky " to represent, individual has one or more so-called risk factors, It is the measurable parameter related to the generation of Alzheimer's.Individual ratio with one or more of these risk factors Individual without these risk factors has the more high likelihood that Alzheimer's occurs.These risk factors include, But it is sudden and violent to be not limited to age, sex, race, diet, medical history, the presence of precursor disease, gene (i.e. hereditary) factor, and environment Dew, and it is well known to those skilled in the art.

In one embodiment, present disclosure provides treats or prevents Alzheimer's or another in subject The method of the progress of one tau lesions, methods described include applying as provided herein at least the one of effective dose to the subject Kind antibody and/or its tau binding fragment.In some embodiments, methods described can reduce dyskinesia, improve motion work( Can, cognitive disorder is reduced, improves cognitive function, or its combination.

In other embodiments, present disclosure provides improves and Alzheimer's or another in subject The method of the related at least one symptom of tau lesions, methods described are included to the subject using effective dose as carried herein At least one antibody and/or its tau binding fragment supplied.

In one embodiment, the method that present disclosure provides delay Alzheimer's progress.In a reality Apply in scheme, the progress of " delay " Alzheimer's represents postpone, hinder, slowing down, postponing, stablizing and/or extension disease Generation.The delay can be different time length, and this depends on history of disease and/or the individual treated.Such as to ability Field technique personnel are it is readily apparent that sufficiently or significantly postpone to include preventing, because this does not occur for individual Disease.In one embodiment, " delay " Alzheimer's occur method be compared with without using methods described, Disease possibility occurrence and/or the method for reducing disease degree are reduced in fixed time limit.This is relatively typically based on using statistics The clinical research of upper significant amount of subject.

Patient, subject or individual include mammal, such as people, ox, horse, dog, cat, pig, and sheep animal.Subject is preferred People, and can be tormented by disease or show symptom at present or be not subjected to disease and tormented or do not show symptom at present.In A Er In the case of the Mo's disease of thatch sea, in fact anyone is in risk or by Alzheimer's, if what he or she lived Long enough.Thus, this method does not need any assessment of subject patient's risk and prophylactically applied to general groups.

In one embodiment, patient herein optionally carries out diagnostic test before treatment.In an implementation In scheme, this method is used for the individual of the known genetic risk with Alzheimer's really.Such individual includes having Those of the relative of the disease are had been subjected to, and its risk those are determined by analysis heredity or biochemical markers.It is right In Alzheimer's risk genetic marker include app gene in mutation, particularly position 717 and position 670 with And the mutation on 671, it is referred to as Hardy and Swedish mutation (referring to Hardy (1997) Trends Neurosci.20: 154-9).Other marks of risk are the mutation in presenilin genes PS1 and PS2, and ApoE4, AD family's history, blood courage Sterol is excessive or atherosclerosis.Can be from the presence identification mesh of the dementia of characteristic, and risks and assumptions described above The preceding individual by Alzheimer's.In addition, can obtain many diagnostic tests be used for identify suffer from AD individual.These bags Include the horizontal measurements of CSF tau and amyloid-β 42.The elevated tau and horizontal expression AD of the amyloid-β 42 reduced Presence.In one embodiment, can by ADRDA (Alzheimer's and related symptom assist) standard diagnostics by The individual of Alzheimer's.In asymptomatic patient, can any age (for example, 10,20,30 years old) start to treat. Typically, however, need not start to treat, until patient reaches 40,50,60 or 70 years old.Treatment generally caused in a period of time Interior multidose turns into required.Can be by various ways known in the art with time monitoring treatment.It is comprehensive in potential Down's , can be before birth by applying therapeutic agent to mother or starting to treat after birth soon in the case of simulator sickness patient.

Any selection standard applied in field that can be by treating AD and related tau lesions completes patient's selection. In one embodiment, standard is those of particular clinical trial measure.In another embodiment, standard is by accreditation Those of therapeutic scheme measure.The combination included with exclusion standard can be applied.The example of inclusive criteria includes, but are not limited to： Based on the possible Alzheimer's of NINCDS/ADRDA standard diagnostics；If MMSE scores confirm AD in the range of 15-26 For slightly to moderate；Magnetic resonance imaging scanning (MRI) result of patient's brain is consistent with AD diagnosis；Pass through suitable imaging side Method, for example, by using (11) C-PBB3 or the radiopharmaceutical based on Lansoprazole measure patient's brain in tau pathology presence； And the age between 50 to 85 years old.

In one embodiment, patient's selection follows the method described in the following：Rollin-Sillaire etc. Reasons that prevent the inclusion of Alzheimer's disease patients in clinical trials.Br J Clin Pharmacol.Apr 2013；75(4):1089-1097.

Some for being used to how to assess treatment and measure effective dose there is provided herein many outcome measurements and primary end of the final point Embodiment.In one embodiment, specify time frame in Primary Outcome measurement include Alzheimer's activity scale- Recognize the Change in Mean of subscale 13 (ADAS-Cog13) score and the activity of Alzheimer's collaborative research-daily life (ADCS-ADL) Change in Mean of score and second level outcome, which measure, includes biomarker in celiolymph (total tau, phosphorylation Tau, A β 1-42 are horizontal) change, the change (assessing structure MRI) of MRI capacity analysis, clinical dementia grade (CDR-SB/ CDR-GS change, the change of psychoneural behavior)：Psychoneural questionnaire (NPI) it is total and field score, the change of cognition： MMSE total scores, and/or security：The incidence that adverse events, serious adverse events and treatment are interrupted.In another embodiment party In case, primary end of the final point include it is following in it is any：The time of death generation, institutionalization, the ability for carrying out ADL Forfeiture, serious dementia, the progression of disease speed that slows down, ADCS-ADL, ADAS-cog score, MMSE scores, cognitive performance, blood plasma CSF biomarkers, ADAS total scores, the quality of life assessed by quality of life Alzheimer's scale, behavior are surveyed Try score, and US FDA ' s clinical dementias grading-box summation.

In one embodiment, treatment Alzheimer's refers to reduction or prevents with the behavior of time, function and recognize Know deterioration.In some embodiments, can be tested by series of standardsization well known by persons skilled in the art any one Kind or it is a variety of come in terms of assessing the behavior of Alzheimer's, function and cognition, the standardized test includes, but are not limited to Neuropsychological test, mini-mentalstate examination, simple cognition inspection, psychoneural questionnaire, Ross dementia measuring scale, western class Tooth language and English neuropsychological measuring scale (SENAS), psychotic behavior evaluation, assessment of function, the test of picture clock, Boston orders Test, Jia Lifuni verbal learnings test, recognize symptom checklist, the Test that works continuously, controlled words and phrases association test, cognition Scale, the d2 tests of notice, Delis-Kaplan perform functions system, dull-witted grading scale, the test of digital vigilance, task Fluency test, finger tapping test, the experiment of Kazakhstan class test, Halstead-Reitan Neuropsychology, Hooper visions Tested tissue, the evaluation of Kaplan Baycrest neuro-cognitives, the short Neuropsychological Assessments of Kaufman, Luria-Nebraska god Through psychological tests, memory measuring scale, fast neuronal filler test, the repeatable examination for evaluating Neuropsychology state Test, Stroop test, sign digit pattern test, tactile property test, theme understand test, Tower of London, trace describe test A Tested with B, speech (word) fluency, and the test of winconsin card sorting.Also use well known by persons skilled in the art is used In depression and anxiety, aphasia, excitement, and the extra test of behavior reference.

In another embodiment, by the raising of the deterioration grade in following at least one evaluation, or do not dislike Change, or reduce to determine the treatment of Alzheimer's：Alzheimer's measuring scale-cognition subscale (ADAS- Cog), clinical dementia grading-box summation (CDR-sb), Alzheimer's collaborative research ADL scale (ADCS- ADL), psychoneural questionnaire (NPI), and Mini-Mental Status assess (MMSE).In some embodiments, treatment causes Deteriorate the reduction of grade in ADAS-cog scores.In other embodiments, treatment causes to deteriorate grade in ADAS-cog scores The reduction of two-five points.

In one embodiment, based on from food and medicine Surveillance Authority of the U.S. available FDA ' s of form renewal “Guidance for Industry,Alzheimer’s Disease:Developing Drugs for the Treatment Of Early Stage Disease, " identify and/or select patient.

In one embodiment, according to National Institute of Neurologic and Communicative Disorders and Stroke-Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA) standard carries out the diagnosis of Alzheimer's in people experimenter.

Periodically using these one or more tests doctor or other medical professions can be suggested on A Erci The progress or degeneration of extra large Mo's disease and related tau lesions and the needs for further treating.The selection of test and treat successfully Determination in the professional knowledge of Alzheimer's field Chinese medicine major personage.Improved in one or more test Point it is the instruction that Alzheimer's seriousness reduces in subject.

Diagnostic method

Because it detects the ability of tau pathologic forms, antibody and its tau binding fragment as described herein be used for it is many its His practical application.The example of such application includes, but are not limited to pathologic tau binding analysis, disease tendency is screened, disease is examined Disconnected, disease prognosis, progression of disease monitoring, the level based on individual type and pathologic tau determine therapeutic strategy, based on pathologic Tau levels and the type related to disease or the possibility of response medicine exploitation therapeutic agent, it is that clinical test is layered PATIENT POPULATION The possibility of therapeutic agent will be responded for therapeutic scheme, and prediction individual.Examined using antibody disclosed herein and tau binding fragments The in-vitro method of the survey pathologic Protein tau related to Alzheimer's and related tau lesions includes, but are not limited to enzyme-linked Immunosorbent assay (ELISA), radiommunoassay (RIA), western blot, immunoprecipitation, immunofluorescence, sandwich assay, And tissue array.

In one embodiment, it is different to can be used for assessment pathologic tau for antibody and its tau binding fragment as described herein Unconventionality expression in normal Tissue distribution, growth course, or exceptional condition, such as the table under Alzheimer's and related tau lesions Reach.In addition, circulation pathologic tau antibody test can be used for identifying that the tau during AD and related tau lesions treatments is turned over Turn.

In the relevant embodiments, the present invention provides Alzheimer's or another tau in diagnosis or screening subject The presence of lesion, or the method that the risk of Alzheimer's or another tau lesions occurs for measure subject, methods described bag Include：

A) make subject, or the cell of subject, tissue, organ, fluid or any other sample with provided herein is have At least one antibody of effect amount and/or the contact of its tau binding fragment；With

B) presence of the measure comprising pathologic tau with antibody and/or the complex of its tau binding fragment, its mesocomplex Presence be diagnosed as Alzheimer's or to pathologic tau exist related another tau lesions.

In some embodiments, antibody and/or its tau binding fragment can be used for internal detection, in vitro, original position, body Outside, in body fluid (for example, blood, serum, urine, blood plasma, cerebrospinal fluid, saliva), or the pathology in cell lysate or supernatant Property tau is to assess the amount of expression and pattern.In one embodiment, antibody and its tau binding fragments are surveyed for in-vivo diagnostic It is fixed, such as in-vivo imaging.In some of these embodiments, using radionuclide (such as¹¹¹In、⁹⁹Tc、¹⁴C、¹³¹I、¹²⁵I or³H) labelled antibody, so as to be located through the presence mark of antibody (and pathologic tau) using immunoscintiography The aim cell or tissue of note.Other marks of detectable mark including the use of analytical technology observable, the technology include, But it is common to be not limited to fluorescence, chemiluminescence, electron spin resonance, ultraviolet/visible absorption spectra, infrared light spectrum, mass spectrum, nuclear-magnetism Shake, magnetic resonance, radiation and electrochemical method.

In one embodiment, antibody and its tau binding fragments can be used for assessing Alzheimer's or another In the method for the therapeutic effect of tau lesions.In one embodiment, methods described is including the use of antibody and its tau bonding pads Section one of be used for monitoring treatment before, in therapeutic process and treatment after pathologic tau presence.In one embodiment, pathology Property tau is horizontal or the reduction instruction of type is to the active response of designated treatment.In another embodiment, pathologic tau is horizontal Or type lacks change or increase instruction treatment and should continued.

In one embodiment, first time point can be selected before prevention or treatment start and can prevented Or treatment starts to select second time point afterwards.Pathologic tau can be measured in each sample obtained from different time points Level simultaneously points out qualitative and/or quantitative differences.Pathologic tau from first part and the measurement of second sample it is horizontal a kind of or The change of a variety of amounts can be associated with prognosis, for determining therapeutic efficiency, and/or for determining entering for disease in subject Exhibition.

It can be promoted by antibody or its tau binding fragment being coupled and (that is, physically being connected) detectable substance such as this The combination of antibody or its tau binding fragment described in text.Detectable substance includes, but are not limited to various enzymes, spoke base, phosphor Material, luminescent material, bioluminescent material, and radioactive material.The example of suitable enzymes includes horseradish peroxidase, alkaline phosphatase Enzyme, beta galactosidase or acetylcholinesterase；The example of suitable spoke base complex includes streptavidin/biotin And avidin/biotin；The example of suitable fluorescent material includes umbelliferone, fluorescein, isosulfocyanic acid fluorescence Element, rhodamine, dichlorotriazine amine fluorescein, dansyl Cl or phycoerythrin；The example of luminescent material includes luminol；Biology The example of luminescent material includes luciferase, luciferin and aequorin, and the example bag of suitable radioactive material Include¹²⁵I、¹³¹I、³⁵S and³H。

Other purposes

Antibody and tau binding fragment as described herein can be used for by standard technique, such as affinity chromatography or immune heavy Shallow lake separates pathologic Protein tau from natural cell derived or from recombinant host cell.In another embodiment, this paper institutes The antibody and tau binding fragments stated can be used for other antibody for identifying binding of pathological tau by competition assay.

In one embodiment, antibody may be used as affine-purified reagent.In one embodiment, using ability Method known to domain by antibody or its tau binding fragment be fixed in solid phase as SEPHADEX.TM. resins or magnetic bead (for example, The magnetic bead of Dynal brands) or filter paper.Fixed antibody contacts with the sample containing tau (or its fragment) to be purified, Ran Houli Holder, the Protein tau knot are cleaned with the suitable solvent that can remove essentially all material (in addition to Protein tau) in sample Close on fixed antibody.Finally, using another suitable solvent, such as glycine buffer, (it discharges pH 5.0 from antibody Protein tau) cleaning holder.

There is also provided for the kit using antibody and its tau binding fragments, such as it is used to detect disease in test sample Rationality tau existing kit.Exemplary kit can include antibody and its tau binding fragments, such as mark or can mark The antibody and compound or reagent of note are used to detect the misfolded proteins in biological sample；For misfolded proteins in determination sample Amount, or the method for presence/missing；Method for the amount and standard of misfolded proteins in comparative sample；And operation instructions.

In some embodiments, the present invention provides medical apparatus, and it includes antibody as provided herein or tau bonding pads Section, wherein described device are suitable for by being contacted selected from following at least one mode or administration of antibodies or tau binding fragments：Intestines Stomach is outer, subcutaneous, intramuscular, intravenous, intra-articular, bronchus is interior, abdomen is interior, intracapsular, cartilage is interior, intracavitary, body cavity are interior, small intracerebral, brain In indoor, intrathecal, colon, neck tube, in stomach, in liver, in cardiac muscle, in bone, in pelvis, in pericardium, in intraperitoneal, pleura, it is preceding Row gland is interior, intrapulmonary, rectum are interior, kidney is interior, retina is interior, backbone is interior, synovial membrane intracavitary, intrathoracic, intrauterine, bladder are interior, internal injury, ball Agent, vagina, rectum, oral cavity, sublingual, intranasal and endermic mode.In one embodiment, device includes syringe (example Such as, prefilled syringe).In another embodiment, device includes sticking patch (patch).In another embodiment, device includes Pump (for example, mini-pump).In another embodiment, device includes inhalator.In another embodiment, device includes spraying Device.

In another embodiment, there is provided for the manufactured goods for the material for treating AD and related tau lesions.Manufactured goods can be with Comprising the humanized antibody wherein contained with fixed dosage and/or the bottle of its tau binding fragment and, optionally packing instruction Book.Bottle can be formed by multiple material, such as glass or plastics, and the plug seal that be able to can be punctured by syringe. For example, bottle can be formal type of glass I vials (for example, 20cc bottles be used for a certain fixed dosage or 50cc it is small Bottle is used for another fixed dosage), there is DAIKYO GREY fluororesins layering plug and 20mm to renovate aluminium lid for it.Manufactured goods are also Including from business and the desired other materials of user's position, including other buffer salts, diluent, filter, pin and injection Device etc..In one embodiment, manufactured goods include two bottles, wherein first bottle contains the people for the fixed dosage specified Source antibody and/or its tau binding fragment, and second bottle contain different fixed dosages humanized antibody and/or Its tau binding fragment.

Antibody can be used for producing anti-id AB.(see, for example, Greenspan＆Bona, FASEB is J.7 (5):437-444,1989；And Nissinoff, J.Immunol.147:2429-2438,1991).Such anti-idiotype resists Body can be used for pharmacokinetics, pharmacodynamics, biodistribution research and utilize facing in the individual of Antybody therapy Bed people-anti-human antibody (HAHA) response studies.For example, anti-id AB specific binding humanization DC8E8 antibody Variable region, and thus can be used for detecting humanization DC8E8 antibody in Pharmacokinetics research and help quantitative controlled Treat people-anti-human antibody (HAHA) in individual.

All publications, patent applications and patents quoted in this specification are expressly incorporated herein by reference, just as each Single publication, patent application or patent is incorporated by reference the same by specifically and individually explanation.Specifically, herein Cited all publications, patent applications and patents are description and the composition that can be openly used in combination with the disclosed invention Purpose with methodology is by reference to being clearly incorporated herein.

Abbreviation is applied to embodiment provided below below.

Expi293 human embryo kidney (HEK)s (HEK293 high density/serum-free) cell

A adenines

Bp base-pairs

DEG C degree Celsius

C cytimidines

MEM minimum essential mediums

DNA DNAs

ELISA enzyme linked immunosorbent assay (ELISA)s

EC50 provides the antibody concentration of maximum half effect

EC80 provides the antibody concentration of 80% ceiling effect

ECD extracellular domains

G grams

G guanines

HRP horseradish peroxidases

IgG immunoglobulin Gs

K G or T (IUPAC treaties)

Min minutes

M A or C (IUPAC treaties)

Nm nanometers

OD optical density

PBS phosphate buffered saline (PBS)s

PCR PCRs

R A or G (IUPAC treaties)

RNA ribonucleic acid

RT room temperatures

The s seconds

S C or G (IUPAC treaties)

T thymidines

TBS trimethylolaminomethane buffered salines

UV ultraviolet lights

V A or C or G (IUPAC treaties)

VH immunoglobulin heavy chain variables area

VK immunoglobulin kappa light chains variable region

W A or T (IUPAC treaties)

Y C or T (IUPAC treaties)

Embodiment

Embodiment 1：The generation of the chimeric version of DC8E8 antibody

Using the RNeasy Mini schemes (Qiagen) separated for total serum IgE total serum IgE is separated from DC8E8 hybridomas. DC8E8 RNA (3 μ g) are inverted according to the scheme of manufacturer using GE Life Sciences the first chain cDNA synthetic agent box Record to produce DC8E8 cDNA.As expanded DC8E8 cDNA by PCR in 3 times individually reaction described in 8.3 parts.Utilize Heavy chain primer M13-MHV6 plus MHCmix and κ light chain PCR primer M13-MKV5plus MKC, use Phusion High- Fidelity PCR Master Mix (Thermo Scientific) PCR expands immunoglobulin heavy chain variable area (VH) cDNA.The result of each PCR reactions is single amplified production, and it is purified and used using QIAquick PCR purification kits M13- forward directions (TGTAAAACGACGGCCAGT (SEQ ID NO:) and M13- reverse primers (CAGGAAACAGCTATGACC 158) (SEQ ID NO:159)) it is sequenced in the two directions.

MHV represents the primer hybridized with the targeting sequencing of murine heavy chain variable region gene, and MHCG is represented and murine constant region The primer of gene recombination.Sequences in italics represents M13 forward directions or M13 reverse sequencing primers.

MKV represents the primer hybridized with the targeting sequencing of mouse kappa light chain variable region gene；MKC is represented and mouse kappa constant region The primer of gene recombination.Coloured part represents M13 forward directions or M13 reverse sequencing primers.By the shared of DC8E8 VK PCR Sequence designations are DC8E8 VK (Fig. 3) and VH shared DNA sequence dna are named as into DC8E8 VH (Fig. 4).The germline analysis of sequence Show that κ light chains are muroid VK8, without somatic mutation (Fig. 5).Heavy chain is muroid VH1, and is shown in CDR ' s and framework Many somatic mutations (Fig. 6).Think that it is important to occur two proline residues in framework 1.These residues existIt is neighbouring residual There is structure function outside base.

It (is mark herein into IgG/ κ carriers that structure chimeric viral vectors, which are needed the variable region routine cloning of amplification, PHuG1, pHuG4 and pHuK, it is commercially available that its is various).Selection produces the clone of correct construct and sequencing.

Utilize DC8E8 VH.pHuG1 and DC8E8 VK.pHuK or DC8E8 VH.pHuG4 and DC8E8 VK.pHuK (each 50 μ g DNA), the Expi293 grown using the reagent cotransfections of ExpiFectamine 293 in Expi293 transfection medias (Invitrogen) suspension cell.Cell grows 10 days in 100ml growth mediums.Pass through routine in conditioned medium ELISA method measures the κ of γ 1 and the κ of γ 4 (chimeric DC8E8 antibody) presence.

The Protein tau binding activity of each chimeric antibody is measured by combining ELISA and by itself and initial mouse DC8E8 antibody Compare.94 hole MaxiSorp flat boards are coated with using the Tau 151-391/4R of the 330ng/mL of 50 μ L aliquots in PBS (Nunc) each hole is simultaneously incubated overnight at 4 DEG C.Using PBS-T (0.1%Tween20) clean-out opening three times.Utilize 250 μ L's Close fresh hole and incubated 1 hour under RT in PBS/0.2%BSA/0.05%Tween20/ holes.Utilize PBS-T (0.1% Tween20) clean-out opening is three times.240uL antibody is added into the hole of the 1st row (if necessary in PBS/0.2%BSA/0.05% Diluted in Tween20)；120 μ L buffer solution (PBS/0.2%BSA/0.05%Tween20) is added in other holes.By 120 μ During L is from the 1st column jump to the adjacent bores of the 2nd row.Program is continueed into the 12nd row using the laboratory sample of a series of 2 times dilutions. 100 μ L/ holes are transferred to assay plate from dilution plate.Flat board incubates 1 hour under RT.Utilize PBS-T clean-out openings 3 times. Goat anti human's Fc peroxidase conjugateds thing is diluted 10000 times (or 10000 times in PBS/0.2%BSA/0.05%Tween20 The anti-mouse of dilution) and add into each hole 100 μ L.Flat board incubates 1 hour under RT and repeats cleaning step.Added per hole 150 μ L substrate (K-Blue) simultaneously incubates 150 minutes under RT.Terminated by the RED STOP solution that 50 μ l are added into each hole Reaction.Optical density is read at 650nm.Two chimeric antibodies are with suitable EC50 value combination Tau 151-391/4R, itself and mouse Class DC8E8 antibody is quite (Fig. 7).Sequence is used for the humanization version for designing DC8E8 antibody.

Embodiment 2：Mouse DC8E8 humanization variants

Select immunoglobulin sequences M65092 as be used for humanized heavy chain's framework (FW) people donor candidate, this be by In its sequence identity higher with DC8E8 variable weight districts (VH) and similitude.The sequence alignment of the two variable regions is visible In Fig. 8.It is the acceptor FW that CDR1,2 and 3 are transplanted to M65092 from DC8E8 VH in next step.Kabat positions 9,21,27,28, 30th, people's residue on 38,48,67,68,70 and 95 is not guarded in M65092 wild type variable weight district (RHA).Due to it Position is (the Kabat CDR's using program Discovery Studio (Accelrys)It is interior, except on position 9 and 21 Pro), these residues are tested with its importance in tau combinations.The step is least predictable in monoclonal antibody human source One of program, and force crucial Framework residues of the identification from parental antibody, it needs to be retained substantially to retain parent The binding property of this antibody, while the possibility immunogenicity of gained humanized antibody is minimized.These non-conservative residues are each From back mutation into DC8E8 mouse equivalent ones, multiple restructuring variable weight district RHA are produced by RHM.(Fig. 8).

For humanization light chain, with method surveyor κ (light) chain as heavy chain class.Initial analysis is found that some possibility Donor candidate, but it is all these prove it is people VK4, it shows weak expression.Analysis is extended to residual including few one The CDR1 of base produces single candidate X72449, and it is than VK4 candidate display and the sequence homology of rodent antibody higher degree. The sequence of the variable κ light chains (VK) of DC8E8 and X72449 variable κ light chains compare.Fig. 9.RKA display restructuring variable lights, its DC8E8 ' s CDRs are transplanted on X72449 frameworks.In optional variant RKB, DC8E8 ' s CDRs are transplanted on X72449 frameworks And the back mutation of Kabat residues 5 is into corresponding DC8E8 light chains mouse residues.

RHA to RHM, RKA and RKB gene are synthesized by GenScript and/or PCR mutagenesis.It is special using GenScript Some software algorithms, the codon preferentially utilized with user's cell by silent mutagenesis codon optimised genes.Then use Each gene of RHA to RHM genes is inserted into human IgG1 and IgG4 expression vectors by the method that this area is generally carried out.With identical Mode RKA and RKB genes are inserted into K light chain expression vectors.Those skilled in the art are known and can pass through commercial sources Obtain the lot of examples of such expression vector.By cloning and sequencing and use QIAGEN Plasmid Miniprep/Maxiprep examinations Agent box prepares expression plasmid DNA.The expression plasmid preparation for encoding all different humanizations and chimeric VH and VK sequences is used to make Expi293 cells are transfected with Invitrogen ' s Expi293 expression systems kit, are cultivated 10 days in serum free medium, The conditioned medium containing each secretory antibody is being collected thereon.When needed, ELISA is passed through using the quantitative conventional method of antibody Measure the concentration of IgG1k and IgG4k antibody in Expi293 conditioned mediums.

Embodiment 3：The property of DC8E8 humanization versions

The combination that Protein tau passes through DC8E8 antibody

As described in example 5 above, by combining ELISA measurements and the binding activity of Tau albumen (151-139/4R).Figure 10 The data of middle display show the combination effect of humanization DC8E8 initial versions.Although all versions are with reference to the initial group Tau albumen, but those antibody containing heavy chain RHB versions seem more preferable in combination.The version of heavy chain contains to all The back mutation of muroid residue on Kabat positions 9,21,27,28,30,38,48,67,68,70 and 95, shows these residues It is one or more be important to retaining full length antibody binding activity.

Other versions of humanized heavy chain are synthesized, it each has single back mutation, and tests and tie by ELISA Close.Shown in Figure 11,12 and 13 and outline the result of IgG4 versions.Antibody version with RHB, RHD and RHE is consistent more Good, it has humanization light chain (RKA and RKB) or chimeric light chain (cDC8E8).Because RHD and RHE versions only contain single mouse 11 muroid back mutations in class back mutation, with version RHB on the contrary, select the two versions as possible leading candidate, It is conjugated with RKA light chain versions (no muroid back mutation)., it is surprising that these back mutations may be enough to recover with The binding affinity of humanized antibody.Combination RHD and RHE mutation (version RHM) do not cause pathology relative to each individually mutation Property tau binding properties significantly improve.

According to the above results, the weight containing two muroid back mutations present in RHD and RHE is produced by direct mutagenesis Chain version (RHM).Figure 14 shows the RHM versions of Tau protein 15 1-391/4R combination humanization DC8E8 antibody, and shows pair Leading candidate AX004 (RHD/RKA) is no better than in the affinity of albumen.

Heat endurance of the humanization candidate antibodies to high temperature

The purpose of the experiment is to compare the heat endurance of humanized antibody.When by higher temperature, 85 are changed to from 30 ° DEG C, 10 minutes, it is cooled to 4 DEG C and is used to combine in ELISA measure with each candidate of EC80 concentration.All antibody tested are seen Get up same stable (Figure 15), and all antibody only become to inactivate at 70 DEG C, but not any to tau binding properties before this Undesired influence.

Humanization candidate antibodies Tm measure

In order to determine leading antibody A X004 (DA), AX005 (EA), AX016 (DB) and AX017 (DE) melting temperature, In 2 step affinity chromatographys and gel filtration system (as described in example 4 above) purifying humanization, chimeric and mouse antibodies and Tested in heat transfer.The hot transfer assay is the microplate combination mensuration for monitoring hot melt solution curve.Methods described uses The dyestuff SYPRO Orange of context-sensitive, its protein binding property increase with protein denaturation.Thus, the protein analyzed After being denatured by thermal induction, the combination of dyestuff increases and reaches maximum when protein is denatured/deployed completely.As temperature funtion Therefore the fluorescence intensity of drafting produces typical sigmoid curve, wherein flex point (half maximum intensity) corresponds to the melting temperature of protein Spend (Tm).Sample incubates 71 circulations with fluorescent dye (Sypro Orange) in qPCR thermal cyclers, each circulation increase by 1 ℃.Chimeric and four humanized antibodies Tm is indicated in figure 16 and confirms the knot obtained in thermal stability determination Fruit：All candidate antibodies have closely similar Tms (69-70 DEG C) and similar to those (70 DEG C) of chimeric antibody, but The slightly lower than Tm (73 DEG C) of mouse antibodies.

The affinity of humanization candidate antibodies

Analyzed using Biacore T200 by SPR and carry out affinity of antibody measure.Anti-human igg (GE Healthcare, Cat.no.BR-1008-39) or anti-muroid IgG (GE Healthcare, cat.no.BR-1008-38) is covalently fixed to CM5 cores Piece (Cat.no.BR-1005-30) up to saturation point and according to the specification of manufacturer.In HBS-EP buffer solutions [0.01M HEPES pH 7.4,0.15M NaCl, 3mM EDTA, 0.005%v/v surfactant P20, GE HEalthcare] in dilute Antibody mouse DC8E8, it is fitted together to DC8E8, AX004, AX005, AX016 and AX017 and is fixed to 144 by affinity combination and arrive 177RU level.In HBS-EP with the 20nM-0.312nM concentration dilutions of doubling dilution restructuring monomer 151-391/4R ( In the PBS of Argon saturations), then injected with 100 μ L/min flow velocity.Binding time is arranged to 150 seconds, and Dissociation time is set For 300 seconds.Tau concentration is rule of thumb selected mutually to obtain curvature in combination.Software kit 2.0 is assessed using BIAcore T200 According to 1:The dynamics of 1 interaction model initial analysis combination/dissociation.

Influence chart is shown in Figure 17-22.Unfortunately, due to dissociating the Bipolar of phase and combining what is additionally combined in phase Some evidences, analytic dynamics are impossible.Shown using steady-state analysis (Steady State Analysis) acquisition Data.Muroid and chimeric DC8E8 show about 10nM affinity.Leading humanized antibody shows similar affinity, from 9mM (RHD/RKB) is changed to about 16nM (RHE/RKA), and it is summarized in Figure 23.These results prompting humanization has succeeded Ground remains the binding activity it is expected in parameter.

The aggregation of humanization candidate antibodies

It is a serious problems in drug development to think aggregation, because aggregation may reduce the yield in production process, Limit the pot-life and because increased immunogenicity causes the effect of reduction in patients.With 0.4mL/ minutes by antibody samples Analyzed in the size exclusion post being expelled in HPLC system and by multi-angle light scattering with determine absolute molal weight and Check aggregation (referring to Figure 24 A, B, C and D).All variants of the mean molecule quantity between 160-169.8kDa do not show aggregation Sign, the average molecular weight range are the desired extents for being used for IgG monomers during the analysis is set.All samples are monodispersed (Mw/Mn<1.05).The mass recovery quality of injection (quality of calculating relative to) between 99.6-99.9%, it shows good Good Protein Recovery and sample appears not to adhere on post or does not contain insoluble aggregation, and it may be by guard column Keep here.Generally speaking, the Notes of Key Data does not have any rendezvous problem in any anti-DC8E8 antibody samples analyzed.

The dissolubility of humanization candidate antibodies

The candidate antibodies and dense with time interval measurement concentrated and purified using solvent adsorption inspissator (MWCO 7500kDa) Degree.All samples are concentrated into 35-41mg/ml, do not precipitate significantly, and are tested in ELISA is combined, the knot Close ELISA and be displayed without sample loss and the combination possibility (Figure 25 and 26) of Tau peptides.Notes of Key Data antibody is up at least It is not easy to precipitate under 35mg/ml concentration.

Frost/thawing of candidate antibodies stress be analyzed

The candidate antibodies sample of purifying then melts 15 minutes at -80 DEG C and carries out within 15 minutes 10 circulations at room temperature. Then sample is analyzed by SEC-MALS to analyze aggregation (Figure 27).Notes of Key Data frost/thawing does not induce the four of all tests The aggregation of individual antibody.Generally speaking, the Notes of Key Data does not have any aggregation to ask in any analyzed anti-DC8E8 antibody samples Topic.

The thermal induction of candidate antibodies stress be analyzed

The candidate antibodies sample heat exposure of purifying is in a) room temperature, b) 20 days at 37 DEG C and c) 50 DEG C.Then SEC- is passed through MALS analyzes sample to analyze aggregation (Figure 28).Generally speaking, the Notes of Key Data is in any analyzed anti-DC8E8 antibody samples There is no any rendezvous problem.

Embodiment 4：Recombinant full-lenght tau isotypes 2N4R and wrong unordered tau 151-391/4R preparation

Restructuring Protein tau is produced from clone τ 40 (Goedert, 1989), it is subcloned into expression plasmid pET-17b (Novagen) expressed in and in bacterium.According to the unordered tau 151-391/ of most long people's tau isotypes 2N4R numbering mistakes 4R and all tau peptides, the 2N4R length are 441 amino acid and therefore also referred to as tau441 (D ' Souza, 2005).Production Raw Protein tau comprises the following steps：A) tau is expressed in bacterium；B) tau purifying is carried out by ion-exchange chromatography；C) pass through Gel filtration carries out tau purifying；D) concentrate and store the tau of separation；

A) bacterial expression people total length tau 2N4R and wrong unordered tau151-391/4R：Expression plasmid is transformed into greatly In enterobacteria (Escherichia coli) production bacterial strain BL21 (DE3).Bacterial cell of the culture containing suitable expression plasmid And such as Sambrook and Russell (2001) " Molecular Cloning:Enter described in A Laboratory Manual " Row induction.At 37 DEG C, there is the 500ml of 100 μ g/ml ampicillins Luria to cultivate and trained in liquid culture medium with 300rpm Support the monoclonal of BL21 (DE3) bacterium and by adding isopropyl-β-D-thiogalactoside (IPTG) to 0.4mM final concentration Induced, the monoclonal of BL21 (DE3) bacterium is converted by the pET-17b plasmids of driving Protein tau or its fragment expression. After 37 DEG C are further incubated for 3 hours, bacterium is collected by being centrifuged 15 minutes with 3,000xg at 4 DEG C.

B) substantially (total length tau is of the same race for the alkaline and neutral Protein tau of (Krajciova etc., 2008) completion as discussed previously Type and tau 151-391/4R) cation-exchange chromatography purifying.After expression, the cracking that bacterial precipitation is resuspended in 10ml buffers Liquid (50mM 1,4- piperazines two ethyl sulfonic acid (PIPES) pH 6.9,50mM sodium chloride (NaCl), 1mM ethylenediamine tetra-acetic acids (EDTA), 5mM dithiothreitol (DTT)s (DTT), 0.1mM phenylmethylsulfonyl fluorides (PMSF), 5% (v/v) glycerine) in, frozen section is in liquid nitrogen and stores up In the presence of -80 DEG C until for purifying Protein tau.Purified for Protein tau, the bacterial suspension fast melt of frost is placed in On ice.50% working cycles are arranged to by using Sonopuls HD 2200, probe TT-13 (Bandelin, Germany), 50W power outputs, 30 seconds, pause 30 seconds, 6 times in ultrasonication bacteria cell wall on ice.By centrifugation (21 at 4 DEG C, 000xg, 15 minutes) clarified lysates and pass through 0.45 μm of film filter filtering supernatant.UseWork station (Amersham Biosciences, Sweden) completes the large scale purification of restructuring Protein tau at 6 DEG C.Flowed with 3ml/ minutes Speed by the lysate of filtering be loaded into using lysis buffer balance 5-ml HiTr ap SP HP posts (GE Healthcare, Uppsala, Sweden) on, and fully cleaned using 60ml lysis buffer until the baseline at 280nm becomes stable.It is logical Cross the Protein tau of gradient (0-30% in 15ml) elution of bound of buffer B (lysis buffer for being supplemented with 1M NaCl).Receive Collect respective 1ml fractions and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).In order to go Except nucleic acid (itself and positively charged Protein tau copurification), collect the fraction containing Protein tau and handed over by second cation Chromatographic step is changed, uses the 5-ml HiTrap SP HP posts (GE of the relatively slow gradient (0-30% in 45ml) with buffer B Healthcare, Uppsala, Sweden) purified.

C) ion exchange layer will be passed through (for all Protein tau all sames) in the final gel filtration step of purifying The Protein tau fraction of collecting that analysis obtains is expelled to the solvent resistant column (Superdex 200 of HiLoad 26/60 with 3ml/ minutes Prep grade column, GE Healthcare) on, it is being respectively used to being supplemented with for alkalescence/neutral or acid Protein tau In 100mM NaCl PIPES or histidine lysis buffer.Collect the Protein tau of elution.

D) in order to carry out the concentration of Protein tau after gel-filtration purified, diluted and converged using 2.5% glycerine of 1.5 volumes The fraction of collection, and HiTrap SP HP posts (alkaline and neutral Protein tau) or HiTrap Q HP posts (acid tau are loaded into again Albumen) on.Then the restructuring Protein tau of concentration is eluted from the post with 1M NaCl discontinuous gradients.Finally, using 5ml HiTrap desalting columns (GE Healthcare) by buffer-exchanged into using argon saturation phosphate buffered saline solution (PBS, 8.09mM disodium hydrogen phosphates (Na₂HPO₄), 1.47mM potassium dihydrogen phosphates (KH₂PO₄), 136.89mM NaCl, 2.7mM sodium chloride (KCl)).Using bicinchoninic acid (BCA) quantification kit (Pierce, USA), mark is used as by the use of bovine serum albumin(BSA) (BSA) Quasi- product complete the protein quantification of purification of samples.Protein tau is divided into work aliquot, it is quick-frozen in liquid nitrogen, and be stored in- 70℃。

Embodiment 5：Chimeric DC8E8 property

It is higher more affine than being shown to total length tau 2N4R to the unordered tau 151-391/4R of mistake that a/ is fitted together to DC8E8 Power.

By ELISA and chimeric DC8E8 pairs of surface plasma resonance (SPR) measure with pathologic tau's and physiological tau Immunoreactive resolving ability.

Tested for ELISA and SPR, on Protein G affinity column from Serum-free Hybridoma supernatant purified mouse DC8E8, it is as follows.By adding the PBS of 0.2 volume and being adjusted doma supernatant to pH 7.5 using the inspection of pH test strips (if necessary, further adjusting pH by adding 4M NaOH), by centrifuging (20,000xg, 4 DEG C, 10 minutes) pre cleaning solution, Filtered by 0.45 μm of film filter, and be loaded on ml protein G sepharose columns (with the flow velocity of 1-0.5ml/ minutes).Utilize 0.1M glycine-HCl, pH 2.7 elutes DC8E8 from post.Neutralize the level of elution immediately using 1M Tris-HCl pH 9.0 Point.The fraction collected for PBS, is concentrated by ultrafiltration, and is stored in -70 DEG C.By measuring the extinction at 280nm Degree, the concentration of antibody is determined using formula c (mg/ml)=A280nm/1.43.

Chimeric DC8E8 is expressed in Expi293 cells and by as above having what is modified a little for mouse DC8E8 descriptions Affinity chromatography and size exclusion chromatography are purified from serum-free conditioned media.Cell culture medium is adjusted to pH 7.5, By centrifuging pre cleaning, filtered by 0.45 μm of film filter, and use Dulbecco PBS as combination and cleaning buffer solution It is loaded on 1ml HiTrap MabSelect SuRe albumin A posts.Utilize the 0.1M citric acids for being supplemented with 150mM NaCl Sodium pH 2.5 elutes chimeric DC8E8 mAb from post.Neutralize the fraction of elution immediately using 1M Tris-HCl pH 9.0. On the Superdex 200pg posts of HiLoad 16/600, using Dulbecco PBS as running buffer, pass through size exclusion The refined fraction collected of analysis.By measuring the absorbance at 280nm, antibody is determined using formula c (mg/ml)=A280nm/1.37 Concentration.

Determined for Salmonella, with 5 μ g/ml in PBS, 50 μ l/ holes are by the unordered tau151-391/4R of mistake or entirely Long tau 2N4R are fixed on elisa plate (Nunc, MediSorp), and are incubated overnight at 37 DEG C.Utilize PBS-0.05%Tween After 20 (at 20-25 DEG C 1 hours) closing is to reduce non-specific binding, flat board and Block buffer (PBS, 0.05%Tween 20) it is small to incubate 1 at 37 DEG C for the continuous antibody dilution (10000ng/ml -0.17ng/ml concentration range) of the three times in 50 μ l/ holes in When.After incubating and cleaning, with 1 in PBS-Tween buffer solutions:The secondary antibody of 4000 dilution peroxidase conjugateds is (anti-human Ig, Pierce, ThermoScientific) and (50 μ/hole) are applied in hole 1 hour at 37 DEG C.It is blue using color after cleaning Solution (colorburst blue solution) (50 μ/hole) is as peroxidase substrate by reaction solution 20 minutes and profit With 50 μ l 2M H₂SO₄Terminated.Using Multiscan MCC/340 ELISA readers (Labsystems) in 450nm Place's measurement absorbance.Think that twice of the reading that light absorption value is at least negative control (PBS) value is positive.

Analysis show chimeric antibody DC8E8 can mistake unordered tau 151-391/4R and physiological tau 2N4R it Between distinguished (Figure 29 A).Chimeric DC8E8 identification physiological tau 2N4R degree identifies that pathologic/mistake is unordered than it Tau 151-391/4R degree is much lower.Importantly, chimeric DC8E8 immunoreactivity and mouse DC8E8's is immune anti- Answering property is quite (Figure 29 B).Chimeric DC8E8 and mouse DC8E8 is with similar EC₅₀Value combine analyzed Protein tau (pathologic and Physiological tau), as shown in Figure 29 C.Generally speaking, these find the chimeric DC8E8 of prompting binding property and initial mouse DC8E8 binding property is suitable.

Surface plasma body resonant vibration (SPR) is used to detect and Quantitative Western knot merga pass directly monitors binding events in real time For determining the thermokinetic parameters of protein complexes (for example, Antibody-antigen complexes).The technology diagnoses conventionally used for characterizing With treatment antibody (see, for example, Karlsson and Larsson, Affinity Measurement Using Surface Plasmon Resonance, Methods in Molecular Biology, volume 248:Antibody Engineering: Methods and Protocols. are by B.K.C.LoHumana Press Inc., Totowa, NJ, (2008) editor).

BIACORE3000 instruments with CM5 sensor chips (Biacore AB, Uppsala) determine for SPR.From Biacore AB obtain amine coupling agent (EDC, NHS, monoethanolamine pH 8.5), P20 detergents and 10mM sodium acetates pH 5.0.These Test at 25 DEG C with 0.005% P20 (PBS-P) as being completed in the PBS pH 7.4 of running buffer.For chimeric DC8E8, Goat anti human Fc polyclonal antibody (SIGMA, Cat.no.I2136) pass through primary in pH 5.0 in two flow chambers Amine is coupled to 5,000RU (response unit) simultaneously, and one in described two flow chambers is used for reference measure.For mouse DC8E8, utilize Anti-TNF-α mouse antibodies (No.Z 0420；DakoCytomation, Glostrup, Denmark) coating analysis Property flow chamber.

In each analysis circulation, captured respectively in analytical flow chamber the chimeric DC8E8 and mouse DC8E8 of purifying with up to To the fixed horizontal of 200-250RU.Binding constant (K is combined in order to determine balance_A) and measure Kinetic rate constant (kON and KOFF), the tau151-391/4R of twice of serial dilution and physiological tau 2N4R (are directed to by it with the flow velocity of 100 μ l/ minutes Measure DC8E8 affinity) or PBS-P be expelled to as control on sensor chip.Described by Myszka (1999) and pass through BIA Assessing software 4.1 (Biacore AB) fitting, dual reference driving force binding number obtains dissociation rate constant and combination according to this respectively Speed constant.Equilibrium association constant K_AObtain as the ratio of association and dissociation speed constant.

In order to which quantitatively chimeric DC8E8 is to the affinity for the Protein tau tested, measure DC8E8 and physiological, four repetitions The combination equilibrium association constant of Protein tau isotype 2N4R and the unordered tau 151-391/4R of pathologic mistake combination (K_A).Two kinds of Protein taus for SPR are prepared as described in example 4 above.Chimeric DC8E8 antibody is in the unordered tau151- of mistake Distinguished (Figure 30) between 391/4R albumen and physiological Protein tau 2N4R.The degree of chimeric DC8E8 resolving ability is even The resolving ability of slightly higher than initial mouse DC8E8 antibody.These results confirm：(1) wrong nothings of the humanization DC8E8 to tau The specificity of the form of sequence, and (the unordered tau of 2DC8E8dui mistakes (that is, disease or pathologic tau) is relative to total length tau The selectivity of (that is, normal or physiological tau).

B/ is fitted together to DC8E8 combination tau peptides, and four are carried in the duplicate block of its each comfortable albumen tau micro-pipe binding domain One in DC8E8 epitopes

Previous results show mouse DC8E8 have four combinations site on people tau or epitope (267-273,298-304, 329-335 and 361-367), it is each respectively positioned at (WO/2013/ in one in the repetition in tau micro-pipe binding domain 041962, Kontsekova etc., 2014).Four any one abilities of epitope are combined in order to test chimeric DC8E8, are passed through EZBiolabs (USA) synthesis purity is higher than 95% tau peptides 256-285,282-311,314-342,352-380.Each peptide includes One of four single DC8E8 epitopes.Chimeric DC8E8 and the binding activity of tau peptides, such as embodiment are measured by Salmonella Described in 5a/.Chimeric DC8E8 combines the MTBR sources peptide (Figure 31 A) of all tests, (figure similar with initial mouse DC8E8 31B).Importantly, highest immunoreactivity shows chimeric and mouse DC8E8 and peptide 282-311, it derives from MTBRII simultaneously And it includes the DC8E8 epitopes in the 298-304 of position.Chimeric antibody and peptide (256-285,314-342, the 352- additionally tested 380) immunoreactivity is slightly or even higher than with initial mouse DC8E8 immunoreactivity, such as EC₅₀(Figure 31 C) shown in value.

C/ is fitted together to DC8E8 and suppresses pathologic tau-tau interactions

External tau filaments are determined for determining whether chimeric antibody has suppression to pathologic tau-tau interactions Effect.The intrinsic property of the measure based on Protein tau, i.e. itself and polyanion, such as the glycosaminoglycan heparin phase of sulphation Through going through the ability of conformation change after interaction.The conformation of this change on one tau molecule further results in itself and another tau The pathologic interaction of molecule, tau-tau complexs are handed over by being formed in the micro-pipe land of the tau molecules of interaction Fork-β-pleated sheet structure and stablize, and finally, formed Alzheimer's sample conjugate spirals fibril (PHF) (Skrabana, R., Sevcik,l.,Novak,M.(2006)。Intrinsically disordered proteins in the neurodegenerative processes:formation of tau protein paired helical filaments and their analysis.Cell Mol Neurobiol 26,1085-1097).Can be by fluorescent dye, as thioflavine T detects the formation of the structure rich in β-pleated sheet (Friedhoff P, Schneider A, Mandelkow EM, Mandelkow E.Rapid assembly of Alzheimer-like paired helical filaments from microtubule- associated protein tau monitored by fluorescence in solution.Biochemistry 37 (28):10223-30(1998))。

Determined for external tau filamentizations, pass through the method purified mouse described in embodiment 5 and chimeric DC8E8. Measure is set in PBS to measure influence that chimeric DC8E8 interacts to pathologic tau-tau (by 0.2 μm of filter mistake Filter), the PBS contains：The wrong unordered tau 151-391/4R of 10 μM (final concentration)；10 μM of heparin (glue from chitterlings The Calciparine/sodium salt of film, >=150IU/mg, dry basis, mean molecule quantity 6000Da, from SIGMA)；With 12.5 μM (final concentrations) Thioflavine T.Each reaction (50 μ l final volumes) is at 37 DEG C in black solid polystyrene plate (384 holes, the Greiner of sealing BioOne incubated 20 hours in).Using fluorescence reader (Fluoroskan Ascent FL (Labsystems)), with 450nm Excitation wavelength, 510nm launch wavelength, and 200ms time of measuring measurement thioflavin T fluorometric.In order to determine chimeric DC8E8 To the inhibitory activity of pathologic tau-tau interactions, 10 μM of final concentrations of addition in 37 DEG C of incubation forward reaction mixtures The chimeric DC8E8 of purifying.Conformation is measured by thioflavin T fluorometric in the case where lacking (" no antibody ") and chimeric antibody being present Tau change and filament amount (Figure 32).Mistake is prevented with the chimeric DC8E8 and mouse DC8E8 of 10 μM of final concentration additions The pathologic conformation change and filament of unordered Protein tau.Chimeric DC8E8 reduces the amount of filament pathologic tau forms extremely Less than 5.9%, mouse reduces the amount of filament pathologic tau forms to less than 4.7%.Data show chimeric antibody with parent Ability suitable DC8E8 prevents the pathologic conformation change and filament of the unordered Protein tau of mistake.

Embodiment 6：The property of DC8E8 humanization version

It is higher that the a/DC8E8 humanization version tau 151-391/4R unordered to mistake compares total length tau 2N4R displayings Affinity.

In order to assess DC8E8 humanization version between pathologic and the immunoreactivity of physiological Protein tau Resolving ability, using ELISA and SPR, as described in example 5 above.It is used to purify chimeric DC8E8's according to embodiment 5 Method purifies DC8E8 all humanization variants, i.e. X004, AX005, AX016, AX017 (in isotype IgG4 and IgG1).

ELISA shows that all humanizations leading AX004, AX005, AX016 and AX017 (IgG4 and IgG1 isotypes) are equal Pathologic tau151-391/4R (Figure 33 A-D can be identified；Figure 34 A-D).Importantly, each humanization DC8E8 variants are to pathology Property tau 151-391/4R combination higher than the combination to physiological tau 2N4R.However, the RHE versions containing heavy chain is leading (AX005 and AX017) seems weaker (Figure 33 F than the leading AX004 and AX016 of the RHD versions containing heavy chain with reference to physiological tau； Figure 34 E).Although AX005 and AX017 combination physiologicals tau is weaker, humanization leading AX004, AX005, AX016 and AX017's The degree of resolving ability is similar with chimeric DC8E8 antibody, such as EC₅₀Shown in value.Generally speaking, the knot of the humanized antibody of test It is suitable with the binding property of chimeric antibody (and parent mouse DC8E8, Figure 29) to close property.

For being tested as described above for the chimeric DC8E8 SPR carried out, the humanization variants of DC8E8 monoclonal antibodies are used With chimeric DC8E8.It is each analysis circulation in, captured respectively in analytical flow chamber DC8E8 purifying humanization version with Reach the fixed horizontal of 200-250RU.In order to which quantitative humanization DC8E8 is to the respective affinity of Protein tau tested, measure The antibody tau 151-391/4R unordered with physiological, four repetition Protein tau isotype 2N4R and pathologic mistake knot The combination equilibrium association constant (KA) of conjunction.All Protein taus for SPR are prepared according to embodiment 4.Each humanization DC8E8 becomes The body tau 151-391/4R unordered to mistake affinity is higher than the affinity to total length tau 2N4R.(Figure 35 A, B).These As a result confirm：(1) humanization DC8E8 is unordered to mistake to the specificity of tau wrong unordered form, and (2) DC8E8 Tau (that is, disease or pathologic tau) relative to total length tau (that is, normal or physiological tau) selectivity.

B/DC8E8 humanization version combination tau peptides, four DC8E8 tables are carried in its each comfortable tau micro-pipe binding domain One in position

The target of the experiment is to determine DC8E8 humanization leading (AX004, AX005, AX016, AX017, isotype IgG4 and isotype IgG1) with reference to tau peptides 256-285,282-311,314-342,352-380 ability.These peptides each wrap Micro-pipe containing tau combines one in the four independent DC8E8 epitopes repeated in (MTBR).DC8E8 people is measured by ELISA Sourceization is leading with the binding activity of tau peptides, as described in example 5 above.The humanized antibody tested respectively is self-bonded all MTBR Peptide (Figure 36 A-D in source；Figure 37 A-D), it is similar (Figure 36 E) with chimeric DC8E8.This is equal for IgG1 and IgG4 isotype versions It is correct.Humanization candidate antibodies show with peptide 282-311 highest immunoreactivity, the peptide from MTBRII and It includes the DC8E8 epitopes in the 298-304 of position, such as EC₅₀(Figure 36 F shown in value；Figure 37 E).Before RHE versions containing heavy chain Lead (AX005 and AX017) and combine other RHD versions of peptide (256-285,314-342,352-380) ratio containing heavy chain tested This weak (Figure 36 F of leading AX004 and AX016；Figure 37 E).In a word, Notes of Key Data humanized antibody AX004 and AX016 and test Peptide immunoreactivity and chimeric DC8E8 with test peptide immunoreactivity it is similar, such as EC₅₀(Figure 36 F) shown in value.

C/DC8E8 humanization version can suppress pathologic tau-tau interactions

In order to determine humanization candidate antibodies (i.e. AX004, AX005, AX016, AX017, isotype IgG4 and isotype IgG1) the influence formed to tau filaments and tau aggregations, external tau filamentizations measure is carried out (as described in example 5 above). All humanizations for purifying DC8E8 as described in example 5 above are leading.The pathology determined for filamentization is prepared according to embodiment 4 Property Protein tau 151-391/4R.

In order to determine the inhibitory activity that humanization candidate antibodies interact to pathologic tau-tau, before 37 DEG C incubate Leading AX004, AX005, AX016, AX017 (isotype of humanization of the purifying of 10 μM of final concentrations is added into reactant mixture IgG4 and isotype IgG1).Surveyed in the case where lacking (" no antibody ") and tested antibody being present by sulphur production Measure conformational change and filament tau amount.As a result show prevents mistake with all humanized antibodies of 10 μM of final concentration additions The pathologic conformation change and filament (Figure 38 A and B) of unordered Protein tau by mistake.The isotype of antibody does not influence humanization and resisted The suppression potential of body.Humanization leading AX004, AX005, AX016 and AX017 (IgG4 and IgG1 isotypes version) reduce filament Change the amount of pathologic tau forms to less than 3%.Notes of Key Data humanized antibody is prevented with the ability suitable with original mouse DC8E8 The only pathologic conformation change and filament of wrong unordered Protein tau.

Embodiment 7：Chimeric DC8E8 and DC8E8 humanization version identification Alzheimer's brain and other tau lesions In pathology

Human brain tissue sample is obtained from Dutch think-tank (on paraffin mass).The cutting cube on slicer.From A Erci Extra large Mo's disease brain (Braak ' s stage VI) and FTDP17 (R406W mutation) hippocampal gyrus entorhinal cortex, become from Corticobasal Property and (8 μm) of the paraffin section of caudate nucleus of stein-leventhal syndrome be used to study.At cold (+4 DEG C) 98% formic acid Reason section 1 minute, is then heat-treated 20 minutes in pressure cooker (2100 Retriever) at 121 DEG C.Histotomy is being closed Incubate 10 minutes in solution (Section block, Aptum) at room temperature, then with primary mouse antibody DC8E8 (1:200)、 Human antibody AX004, AX005, AX016, AX017 and chimeric DC8E8 (all 1:1000) it is incubated overnight.Then, section and biology It is small that the secondary antibody (Vectastain Elite ABC Kit, Vector Laboratories) of elementization incubates 1 at room temperature When, then react 60 minutes (Vectastain Elite ABC with Avidin-biotin Peroxidase Complex Kit, Vector Laboratories), both of which is under room temperature (25 DEG C).Utilize peroxidase substrate kit (Vector VIP, Vector laboratories, Ca, USA) make immune response visible and utilize methyl green (Vector Laboratories) redyed.Immunoreactive assessment is carried out in the case where optical microscopy is amplified with x100-400.In cell The form details of tau immuno positives damage are defined on the basis of positioning and staining pattern.It is digital using Olympus DP50 are equipped with The Olympus BX51 microscopes (Olympus Optical Co., Ltd., Tokyo, Japan) of camera obtain digital picture.

Alzheimer's, stein-leventhal syndrome, corticobasal degeneration and the human brain of FTDP-17 patient are exempted from Epidemic disease histochemical stain discloses chimeric DC8E8 antibody displays and mouse DC8E8 identicals staining pattern (Figure 39-46, A, B).I Compare chimeric antibody and humanization DC8E8 antibody-AX004, AX005, AX016 and AX017 immunohistochemical staining. In general, humanized antibody AX004 and AX016 (IgG1 and IgG4) show the dye very similar with mouse or chimeric DC8E8 Color pattern.Substantial amounts of neuron entanglement, neuropil filament and neuritis in humanized antibody AX004 and AX016 identification people's AD brains Spot (Fig. 9 C, E；40C, E).In people, the FTDP-17 cases for carrying mutation on Protein tau at R406W, AX004 and AX016 are exempted from Epidemic disease mark is smelt to tangle and neuropil filament (Figure 41 C, E with the neuron in cortex of temporal lobe；42C, E).In corticobasal degeneration In, neuroglia tau pathology (Figure 43 C, E in AX004 and AX016 dyeing caudate nucleus；44C, E).The property fiber crops on progressive core In numbness, AX004 and AX016 identify substantial amounts of oligodendroglia conveyor screw and astroglia patch (Figure 45 C, E；46C, E).On the contrary, AX005 and AX017 is in Alzheimer's (Figure 39 D, F；40D, F) in, in FTDP-17 (Figure 41 D, F), Corticobasal degeneration (Figure 43 D, F；44D, F) in and in stein-leventhal syndrome (Figure 45 D, F；46D, F) in displaying reduce Immunostaining.It is interesting that AX005 and AX017 isotype IgG4 do not dyed in FTDP17 pathological structures (Figure 42 D, F).Generally speaking, humanized antibody AX004 and AX016 and DC8E8 has identical staining pattern.

Claims

1. the anti-tau antibody of humanization or its tau binding fragment, wherein the antibody or binding fragment include：

Respectively CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO.1,2,3 weight chain variable district are included, and is exempted from from people The framework (SEQ ID NO.71) of epidemic disease globulin M 65092；

Respectively CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO.4,5,6 light chain variable district are included, and is exempted from from people The framework (SEQ ID NO.65) of epidemic disease globulin X 72449；With

Each from the heavy chain and constant region of light chain of human immunoglobulin(HIg)；And wherein described heavy chain framework selected from 9, 21st, one or more of 27,28,30,38,48,67,68,70 and 95 position is substituted；The light chain framework it is unsubstituted or It is substituted at 5；And wherein described position is according to Kabat.

2. the antibody or binding fragment of claim 1, wherein heavy chain 9 are occupied by P, 21 occupied by P, 27 occupied by Y, 28 Position is occupied by I, 30 occupied by T, 38 occupied by K, 48 occupied by I, 67 occupied by K, 68 occupied by A, 70 by L Occupy, and/or 95 are occupied by F.

3. the antibody or binding fragment of any one of claim 1 and 2, wherein light chain 5 are occupied by S.

4. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is selected from SEQ ID NO.13-25 (RHA to RHM)；And the sequence of the light chain variable district is SEQ ID NO.26 (RKA).

5. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is selected from SEQ ID NO.13-25 (RHA to RHM)；And the sequence of the light chain variable district is SEQ ID NO.27 (RKB).

6. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.13, and institute The sequence for stating light chain variable district is SEQ ID NO.26.

7. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.14, and institute The sequence for stating light chain variable district is SEQ ID NO.26.

8. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.15, and institute The sequence for stating light chain variable district is SEQ ID NO.26.

9. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.16, and institute The sequence for stating light chain variable district is SEQ ID NO.26.

10. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.17, and The sequence of the light chain variable district is SEQ ID NO.26.

11. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.18, and The sequence of the light chain variable district is SEQ ID NO.26.

12. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.19, and The sequence of the light chain variable district is SEQ ID NO.26.

13. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.20, and The sequence of the light chain variable district is SEQ ID NO.26.

14. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.21, and The sequence of the light chain variable district is SEQ ID NO.26.

15. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.22, and The sequence of the light chain variable district is SEQ ID NO.26.

16. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.23, and The sequence of the light chain variable district is SEQ ID NO.26.

17. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.24, and The sequence of the light chain variable district is SEQ ID NO.26.

18. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.25, and The sequence of the light chain variable district is SEQ ID NO.26.

19. claim 6-18 antibody or binding fragment, wherein the antibody is IgG1 isotypes.

20. claim 6-18 antibody or binding fragment, wherein the antibody is IgG4 isotypes.

21. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.13, and The sequence of the light chain variable district is SEQ ID NO.27.

22. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.14, and The sequence of the light chain variable district is SEQ ID NO.27.

23. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.15, and The sequence of the light chain variable district is SEQ ID NO.27.

24. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.16, and The sequence of the light chain variable district is SEQ ID NO.27.

25. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.17, and The sequence of the light chain variable district is SEQ ID NO.27.

26. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.18, and The sequence of the light chain variable district is SEQ ID NO.27.

27. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.19, and The sequence of the light chain variable district is SEQ ID NO.27.

28. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.20, and The sequence of the light chain variable district is SEQ ID NO.27.

29. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.21, and The sequence of the light chain variable district is SEQ ID NO.27.

30. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.22, and The sequence of the light chain variable district is SEQ ID NO.27.

31. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.23, and The sequence of the light chain variable district is SEQ ID NO.27.

32. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.24, and The sequence of the light chain variable district is SEQ ID NO.27.

33. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.25, and The sequence of the light chain variable district is SEQ ID NO.27.

34. claim 21-33 antibody or binding fragment, wherein the antibody is IgG1 isotypes.

35. claim 21-33 antibody or binding fragment, wherein the antibody is IgG4 isotypes.

36. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.14, it is described The sequence of light chain variable district is SEQ ID NO.26, and the antibody is IgG1 isotypes.

37. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.16, it is described The sequence of light chain variable district is SEQ ID NO.26, and the antibody is IgG1 isotypes.

38. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.17, it is described The sequence of light chain variable district is SEQ ID NO.26, and the antibody is IgG1 isotypes.

39. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.25, it is described The sequence of light chain variable district is SEQ ID NO.26, and the antibody is IgG1 isotypes.

40. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.16, it is described The sequence of light chain variable district is SEQ ID NO.27, and the antibody is IgG1 isotypes.

41. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.17, it is described The sequence of light chain variable district is SEQ ID NO.27, and the antibody is IgG1 isotypes.

42. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.16, it is described The sequence of light chain variable district is SEQ ID NO.27, and the antibody is IgG4 isotypes.

43. the antibody or binding fragment of claim 1, wherein the sequence of the weight chain variable district is SEQ ID NO.17, it is described The sequence of light chain variable district is SEQ ID NO.27, and the antibody is IgG4 isotypes.

44. the anti-tau antibody of humanization or its tau binding fragment, wherein the antibody or binding fragment include：

A. respectively CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO.1,2 and 3 weight chain variable district are included, and is come from The framework (SEQ ID NO.71) of human immunoglobulin M 65092；With

B. respectively CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO.4,5,6 light chain variable district are included, and from people The framework (SEQ ID NO.65) of IgX 72449；With

C. heavy chain and constant region of light chain from human immunoglobulin(HIg).

45. anti-tau antibody or its tau binding fragment, wherein the antibody or binding fragment include：

A. there is the heavy chain of any one of SEQ ID NO.28-40 and 42-55 amino acid sequence；With

B. there is the light chain of any one of SEQ ID NO.57 and 58 amino acid sequence.

46. according to any one of claim 1-45 antibody or its tau binding fragment, wherein the antibody or binding fragment knot Conjunction is selected from HQPGGG (SEQ ID NO:148)、HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) extremely A few epitope, preferably two, all more preferably three.

47. antibody or its tau binding fragment, it is included：

A. comprising SEQ ID NO.1,2,3 CDR-H1, CDR-H2 and CDR-H3 and with SEQ ID NO:13、SEQ ID 14、 SEQ ID 15、SEQ ID 16、SEQ ID 17、SEQ ID 18、SEQ ID 19、SEQ ID 20、SEQ ID 21、SEQ ID 22nd, at least 90%, preferably at least 95% of any one of SEQ ID 23RHK, SEQ ID 24, SEQ ID 25, more preferably at least 98% identical weight chain variable district；With

B. respectively comprising SEQ ID NO.4,5,6 CDR-L1, CDR-L2 and CDR-L3 and with SEQ ID NO:26 at least 90%, preferably at least 95%, more preferably at least 98% identical light chain variable district, wherein the antibody binding is selected from HQPGGG (SEQ ID NO:148)、HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) epitope, preferably two, More preferably all three.

48. antibody or its tau binding fragment, it is included：

A. comprising SEQ ID NO.1,2 and 3 CDR-H1, CDR-H2 and CDR-H3 and with SEQ ID NO:13、SEQ ID 14、SEQ ID 15、SEQ ID 16、SEQ ID 17、SEQ ID 18、SEQ ID 19、SEQ ID 20、SEQ ID 21、SEQ Any one of ID 22, SEQ ID 23RHK, SEQ ID 24, SEQ ID 25 at least 90%, preferably at least 95%, more preferably At least 98% identical weight chain variable district；

B. and comprising respectively CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO.4,5,6 and with SEQ ID NO:27 to Few 90%, preferably at least 95%, more preferably at least 98% identical maturation light chain variable district, wherein the antibody binding is selected from HQPGGG(SEQ ID NO:148)、HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) epitope, preferably Two, all more preferably three.

49. any one of claim 1-48 antibody or binding fragment, wherein the binding fragment is Fab, Fab', F (ab') 2、Fd、scFv、(scFv)₂Or scFv-Fc.

50. any one of claim 1-49 antibody or binding fragment, wherein the antibody or binding fragment be IgG1, IgG2, IgG3 or IgG4 isotype.

51. any one of claim 1-49 antibody or binding fragment, wherein the antibody or binding fragment are IgG1 of the same race Type.

52. any one of claim 1-49 antibody or binding fragment, wherein the antibody or binding fragment are IgG4 of the same race Type.

53. any one of claim 1-52 antibody or binding fragment, wherein the antibody or binding fragment are glycosylated.

54. any one of claim 1-53 antibody or binding fragment, wherein the antibody or binding fragment are with least 5x10^-7 Affinity (K_D) combined with Tau 151-391/4R.

55. any one of claim 1-54 antibody or binding fragment, wherein the antibody or binding fragment are with passing through preservation American type culture collection hybridoma PTA-11994 secrete DC8E8 antibody compare with least 80% it is affine Power, preferably substantially identical binding affinity, or more preferably more preferable affinity combination tau.

56. any one of claim 1-55 antibody or binding fragment, wherein the antibody by American Type culture with being protected The DC8E8 antibody competition combination tau of the hybridoma PTA-11994 secretions of Tibetan center preservation, selected from HQPGGG (SEQ ID NO: 148)、HVPGGG(SEQ ID NO:And HKPGGG (SEQ ID NO 149):150) competed at epitope, preferably two, more preferably All three.

57. any one of claim 1-56 antibody or binding fragment, wherein the antibody is caused by restructuring.

58. the antibody or binding fragment of claim 57, wherein the antibody is the weight in Chinese hamster ovary (CHO) cell line Caused by group.

59. any one of claim 1-58 antibody or binding fragment, wherein the antibody or binding fragment contain through modification To change effector function, half-life period, proteolysis and/or glycosylated Fc areas.

60. any one of claim 1-59 antibody or binding fragment, wherein the antibody or binding fragment are modified to adjust Select from antibody dependent cellular cytotoxicity, complement-dependent cytotoxicity, serum half-life, bio distribution and with Fc by One or more functional characters of the combination of body.

61. any one of claim 1-60 antibody, wherein the antibody has the thermostabilization for being equal to or more than 69 DEG C warm-natured Degree.

62. pharmaceutical composition, it is comprising the antibody or binding fragment according to any one of claim 1-61 and can pharmaceutically connect Carrier, diluent, excipient or the stabilizer received.

63. the composition of claim 62, wherein the composition includes the freeze-dried powder of the antibody or binding fragment.

64. the composition any one of claim 62 and 63, wherein the composition is formulated for infusion or subcutaneous Using.

65. the composition any one of claim 62-64, it further includes second therapeutic agent.

66. the composition of claim 65, wherein the second therapeutic agent and the antibody or binding fragment are chemically conjugated.

67. the composition of claim 65, wherein the second therapeutic agent is used to prevent and/or treat AD.

68. according to any one of claim 65-67 composition, wherein the second therapeutic agent is selected from tau peptides, β-amylaceous Protein peptides (such as N- ends amyloid-beta-peptide), it may or may not be conjugated with other compounds, the diphtheria poison of such as mutation Element；Other anti-tau antibody, for the antibody of amyloid beta, as bapineuzumab, solaneuzumab, Gantenerumab, crenezumab and IVIG immunoglobulin, other immunotherapies of A beta oligomers are targetted, prevent tau high The compound of phosphorylation (hyperphosphorylation) is spent, and being actively and passively immunized for other targeting tau aggregations is treated Method；And its any pharmaceutically acceptable salt.

69. claim 65-67 composition, wherein the second therapeutic agent is selected from amyloid-beta peptide aggregation inhibitor (example Such as, Tramiprosate), inhibitors of gamma-secretase (such as semagacestat) and gamma secretase modulators (tarenflurbil)；And its any pharmaceutically acceptable salt.

70. claim 65-67 composition, wherein the second therapeutic agent is selected from acetylcholinesteraseinhibitors inhibitors (for example, more Donepezil, profit cut down the bright of this, galanthamine, Tacrine, nutritious supplementary pharmaceutical), N-methyl-D-aspartate (NMDA) receptor antagonist Agent (such as Memantine), DNA repair inhibitors (for example, pirenzepine or its metabolin), transition metal chelator, growth because Son, hormone, nonsteroid anti-inflammatory drugs (NSAID), antioxidant, lipid lowering agent, selective phosphodiesterase inhibitors, tau aggregations Inhibitor, kinases inhibitor, resist mitochondria dysfunction Pharmacological inhibitors, neurotrophin, heat shock protein suppress Agent, lipoprotein associated phospholipase A₂Inhibitor, Memantine, anti-apoptotic compound, metal-chelator, DNA repair inhibitors, 3- ammonia Base -1- propane sulfonic acid (3APS), 1,3- third disulfonic acid (1,3PDS), secretion zymoexciter, beta-secretase inhibitor, gamma-secretase Inhibitor, beta-amyloid peptide, amyloid beta antibody, neurotransmitter, β-lamella disrupting agent (beta-sheet Breaker), anti-inflammatory molecular；And its any pharmaceutically acceptable salt.

71. claim 65-67 composition, wherein the second therapeutic agent is selected from：Change described in WO2004/058258 Compound (referring particularly to page 16 and 17), it includes curative drug target (the 36-39 pages), alkanesulfonic acid and alkanol sulfuric acid (alkanolsulfuric acid) (the 39-51 pages), anticholinesterase (the 51-56 pages), nmda receptor antagonist ( 56-58 pages), estrogen (the 58-59 pages), nonsteroid anti-inflammatory drugs (the 60-61 pages), antioxidant (the 61-62 pages), peroxide Compound proliferator activated receptor (PPAR) activator (the 63-67 pages), cholesterol reducing agent (the 68-75 pages)；Amyloid Inhibitor (the 75-77 pages), amyloid form inhibitor (the 77-78 pages), metal-chelator (the 78-79 pages), anti-essence Bioactive substance availability in the sick medicine of god and antidepressants (the 80-82 pages), nutritious supplementary pharmaceutical (the 83-89 pages) and increase brain Compound (see the 89-93 pages) and prodrug (page 93 and 94)；And its any pharmaceutically acceptable salt.

72. diagnostic reagent, it includes the antibody or binding fragment and carrier, dilution according to any one of claim 1-61 Agent, excipient or stabilizer.

73. the immunoconjugates with formula (A)-(L)-(C), wherein：(A) be any one of claim 1-61 antibody or Its binding fragment；(L) it is joint；(C) is reagent；And wherein described joint (L) connection (A) to (C).

74. the immunoconjugates of claim 73, wherein (C) is therapeutic agent, developer, detectable agent or diagnosticum.

75. the immunoconjugates of claim 74, wherein (C) is therapeutic agent.

76. nucleic acid molecules, it encodes any one of claim 1-61 antibody or its binding fragment.

77. nucleic acid molecules, it includes the weight chain variable district (HCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the HCVR or its fragment include：(i) framework (SEQ ID NO.71) of human immunoglobulin M 65092 is derived from, (ii) the HCVR CDR1 of the amino acid sequence comprising SEQ ID NO.1, (iii) include SEQ ID NO.2 amino acid sequence HCVR CDR2, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its piece Section includes and SEQ ID NO:13 to any one of SEQ ID NO.25 amino acid sequence at least 98% identical amino acid sequence Row.

78. nucleic acid molecules, it includes the weight chain variable district (HCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the HCVR or its fragment include：(i) framework (SEQ ID NO.71) of human immunoglobulin M 65092 is derived from, (ii) the HCVR CDR1 of the amino acid sequence comprising SEQ ID NO.1, (iii) include SEQ ID NO.2 amino acid sequence HCVR CDR2, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its piece Section includes and SEQ ID NO:14 amino acid sequence at least 98% identical amino acid sequence.

79. nucleic acid molecules, it includes the weight chain variable district (HCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the HCVR or its fragment include：(i) framework (SEQ ID NO.71) of human immunoglobulin M 65092 is derived from, (ii) the HCVR CDR1 of the amino acid sequence comprising SEQ ID NO.1, (iii) include SEQ ID NO.2 amino acid sequence HCVR CDR2, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its piece Section includes and SEQ ID NO:16 amino acid sequence at least 98% identical amino acid sequence.

80. nucleic acid molecules, it includes the weight chain variable district (HCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the HCVR or its fragment include：(i) framework (SEQ ID NO.71) of human immunoglobulin M 65092 is derived from, (ii) the HCVR CDR1 of the amino acid sequence comprising SEQ ID NO.1, (iii) include SEQ ID NO.2 amino acid sequence HCVR CDR2, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its piece Section includes and SEQ ID NO:17 amino acid sequence at least 98% identical amino acid sequence.

81. nucleic acid molecules, it includes the weight chain variable district (HCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the HCVR or its fragment include：(i) framework (SEQ ID NO.71) of human immunoglobulin M 65092 is derived from, (ii) the HCVR CDR1 of the amino acid sequence comprising SEQ ID NO.1, (iii) include SEQ ID NO.2 amino acid sequence HCVR CDR2, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence, and wherein described HCVR or its piece Section includes and SEQ ID NO:25 amino acid sequence at least 98% identical amino acid sequence.

82. any one of claim 77-81 nucleic acid molecules, wherein the antibody or its fragment include and further include IgG1 The heavy chain of constant region.

83. the nucleic acid molecules of claim 78, wherein the antibody or its fragment include the weight for further including IgG1 constant regions Chain.

84. the nucleic acid molecules of claim 79, wherein the antibody or its fragment include the weight for further including IgG1 constant regions Chain.

85. the nucleic acid molecules of claim 80, wherein the antibody or its fragment include the weight for further including IgG1 constant regions Chain.

86. the nucleic acid molecules of claim 81, wherein the antibody or its fragment include the weight for further including IgG1 constant regions Chain.

87. the nucleic acid molecules of claim 79, wherein the antibody or its fragment include the weight for further including IgG4 constant regions Chain.

88. the nucleic acid molecules of claim 80, wherein the antibody or its fragment include the weight for further including IgG4 constant regions Chain.

89. nucleic acid molecules, it includes the light chain variable district (LCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65) is derived from, (ii) the LCVR CDR1 of the amino acid sequence comprising SEQ ID NO.4, (iii) include SEQ ID NO.5 amino acid sequence LCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its piece Section includes and SEQ ID NO:26, RKA amino acid sequence at least 98% identical amino acid sequence.

90. nucleic acid molecules, it includes the light chain variable district (LCVR) for encoding anti-tau antibody or the nucleotides of its tau binding fragment Sequence, wherein the LCVR or its fragment include：(i) human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65) is derived from, (ii) the LCVR CDR1 of the amino acid sequence comprising SEQ ID NO.4, (iii) include SEQ ID NO.5 amino acid sequence LCVR CDR2, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence, and wherein described LCVR or its piece Section includes and SEQ ID NO:27 amino acid sequence at least 98% identical amino acid sequence.

91. the nucleic acid molecules of claim 89, wherein the antibody or its fragment include and further include the light of people's κ constant regions Chain.

92. the nucleic acid molecules of claim 90, wherein the antibody or its fragment include and further include the light of people's κ constant regions Chain.

93. the nucleic acid molecules of claim 77, wherein the nucleic acid sequence encoding SEQ ID NO.13 to any one of 25 HCVR。

94. the nucleic acid molecules of claim 77, wherein the HCVR of the nucleic acid sequence encoding SEQ ID NO.14.

95. the nucleic acid molecules of claim 77, wherein the HCVR of the nucleic acid sequence encoding SEQ ID NO.16.

96. the nucleic acid molecules of claim 77, wherein the HCVR of the nucleic acid sequence encoding SEQ ID NO.17.

97. the nucleic acid molecules of claim 77, wherein the HCVR of the nucleic acid sequence encoding SEQ ID NO.25.

98. the nucleic acid molecules of any one of claim 89 and 90, wherein the nucleic acid sequence encoding SEQ ID NO.26 and 27 Any one of LCVR.

99. the nucleic acid molecules of claim 77, its include under strict conditions with SEQ ID NO:96-108,111-123 and The nucleotide sequence of any of 127-139 complementary strand thereof, wherein the stringent hybridization condition is included in 5xSSPE, 1% Hybridize in SDS, 1xDenhardts solution at 65 DEG C and cleaned in 2xSSC, 1%SDS, and then used at 65 DEG C 0.2xSSC is cleaned.

100. the nucleic acid molecules of claim 77, it includes the complementation with SEQ ID NO.97,112 or 128 under strict conditions The nucleotide sequence of chain hybridization, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS, in 1xDenhardts solution Hybridize at 65 DEG C and cleaned in 2xSSC, 1%SDS, and then cleaned at 65 DEG C using 0.2xSSC.

101. the nucleic acid molecules of claim 77, it includes the complementation with SEQ ID NO.99,114 or 130 under strict conditions The nucleotide sequence of chain hybridization, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS, in 1xDenhardts solution Hybridize at 65 DEG C and cleaned in 2xSSC, 1%SDS, and then cleaned at 65 DEG C using 0.2xSSC.

102. the nucleic acid molecules of claim 77, it includes the complementation with SEQ ID NO.100,115 or 131 under strict conditions The nucleotide sequence of chain hybridization, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS, in 1xDenhardts solution Hybridize at 65 DEG C and cleaned in 2xSSC, 1%SDS, and then cleaned at 65 DEG C using 0.2xSSC.

103. the nucleic acid molecules of claim 77, it includes the complementation with SEQ ID NO.108,123 or 139 under strict conditions The nucleotide sequence of chain hybridization, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS, in 1xDenhardts solution Hybridize at 65 DEG C and cleaned in 2xSSC, 1%SDS, and then cleaned at 65 DEG C using 0.2xSSC.

104. the nucleic acid molecules of claim 89, its include under strict conditions with SEQ ID NO:109 complementary strand thereof Nucleotide sequence, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS is miscellaneous at 65 DEG C in 1xDenhardts solution Hand over and cleaned in 2xSSC, 1%SDS, and then cleaned at 65 DEG C using 0.2xSSC.

105. the nucleic acid molecules of claim 76, its include under strict conditions with SEQ ID NO:110 complementary strand thereof Nucleotide sequence, wherein the stringent hybridization condition is included in 5xSSPE, 1%SDS is miscellaneous at 65 DEG C in 1xDenhardts solution Hand over and cleaned in 2xSSC, 1%SDS, and then cleaned at 65 DEG C using 0.2xSSC.

106. nucleic acid molecules group, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein first nucleic acid molecules are power Profit requires 77 nucleic acid molecules, and wherein described second nucleic acid molecules include LCVR or its tau combination for encoding anti-tau antibody The nucleotide sequence of fragment, wherein the LCVR or its fragment include：(i) it is derived from human immunoglobulin(HIg) X72449 framework (SEQ ID NO.65), (ii) includes the LCVR CDR1 of SEQ ID NO.4 amino acid sequence, and (iii) includes SEQ ID NO.5 ammonia The LCVR CDR2 of base acid sequence, and (iv) include the LCVR CDR3 of SEQ ID NO.6 amino acid sequence.

107. nucleic acid molecules group, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein first nucleic acid molecules are power Profit requires 89 nucleic acid molecules, and wherein described second nucleic acid molecules include HCVR or its tau combination for encoding anti-tau antibody The nucleotide sequence of fragment, wherein the HCVR or its fragment include：(i) it is derived from framework (the SEQ of human immunoglobulin M 65092 ID NO.71), (ii) includes the HCVR CDR1 of SEQ ID NO.1 amino acid sequence, and (iii) includes SEQ ID NO.2 ammonia The HCVR CDR2 of base acid sequence, and (iv) include the HCVR CDR3 of SEQ ID NO.3 amino acid sequence.

108. the nucleic acid molecules group of claim 106, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein described first Nucleic acid molecules include the coding SEQ ID NO.14 HCVR of anti-tau antibody or the nucleotide sequence of its tau binding fragment, and Second nucleic acid molecules include coding SEQ ID NO.26 LCVR or the nucleotide sequence of its tau binding fragment.

109. the nucleic acid molecules group of claim 106, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein described first Nucleic acid molecules include the coding SEQ ID NO.16 HCVR of anti-tau antibody or the nucleotide sequence of its tau binding fragment, and Second nucleic acid molecules include coding SEQ ID NO.26 LCVR or the nucleotide sequence of its tau binding fragment.

110. the nucleic acid molecules group of claim 106, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein described first Nucleic acid molecules include the coding SEQ ID NO.17 HCVR of anti-tau antibody or the nucleotide sequence of its tau binding fragment, and Second nucleic acid molecules include coding SEQ ID NO.26 LCVR or the nucleotide sequence of its tau binding fragment.

111. the nucleic acid molecules group of claim 106, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein described first Nucleic acid molecules include the coding SEQ ID NO.17 HCVR of anti-tau antibody or the nucleotide sequence of its tau binding fragment, and Second nucleic acid molecules include coding SEQ ID NO.27 LCVR or the nucleotide sequence of its tau binding fragment.

112. the nucleic acid molecules group of claim 106, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein described first Nucleic acid molecules include the coding SEQ ID NO.25 HCVR of anti-tau antibody or the nucleotide sequence of its tau binding fragment, and Second nucleic acid molecules include coding SEQ ID NO.26 LCVR or the nucleotide sequence of its tau binding fragment.

113. the nucleic acid molecules group of claim 106, it includes the first nucleic acid molecules and the second nucleic acid molecules, wherein described first Nucleic acid molecules include the coding SEQ ID NO.16 HCVR of anti-tau antibody or the nucleotide sequence of its tau binding fragment, and Second nucleic acid molecules include coding SEQ ID NO.27 LCVR or the nucleotide sequence of its tau binding fragment.

114. carrier, it includes the nucleic acid according to any one of claim 77-105.

115. carrier, it includes the nucleic acid group according to any one of claim 106-111.

116. carrier, it includes the nucleic acid encoded according to any one of claim 1-61 antibody or the heavy chain of binding fragment.

117. carrier, it includes the nucleic acid encoded according to any one of claim 1-61 antibody or the light chain of binding fragment.

118. host cell, it includes any one of claim 114-117 carrier.

119. the host cell of claim 118, wherein the host cell is protokaryon.

120. the host cell of claim 118, wherein the host cell is eucaryon.

121. host cell, it includes any one of claim 106-113 nucleic acid molecules group.

122. producing the method for the antibody or its tau binding fragment with reference to people tau, it includes cultivating in claim 116-119 The host cell of any one so that express the nucleic acid and produce the antibody, its tau binding fragments, heavy chain or light chain.

123. suffer from or be easy to treat in the subject with Alzheimer's or another tau lesions suffering from, suspecting The method of Alzheimer's or another tau lesions, it includes applying (1) of therapeutically effective amount to the subject and included According to any one of claim 1-61 antibody or the composition of tau binding fragments, or (2) according in claim 62-71 The composition of any one.

124. tau aggregations are promoted to suffer from or be easy to suffer from Alzheimer's or another tau lesions from suffering from, suspects The method removed in the brain of subject, it includes applying (1) of therapeutically effective amount to subject and included according to claim 1-61 Any one of antibody or tau binding fragments composition, or (2) according to any one of claim 62-71 composition.

125. suffer from or be easy to slow down in the subject with Alzheimer's or another tau lesions suffering from, suspecting The method of the progress of AD or another tau lesions, it includes including using (1) of therapeutically effective amount to subject wants according to right Any one of 1-61 antibody or the composition of tau binding fragments, or (2) are asked according to any one of claim 62-71 group Compound.

126. suffer from or be easy to improve in the subject with Alzheimer's or another tau lesions suffering from, suspecting The method of the symptom of AD or relative, it includes applying (1) of therapeutically effective amount to subject and included according to claim 1-61 Any one of antibody or tau binding fragments composition, or (2) according to any one of claim 62-71 composition.

127. suffer from, suspect suffer from or be easy to treatment in the subject with Alzheimer's or another tau lesions, Prevention or the method for reversing cognitive (cognitive), it includes applying (1) of therapeutically effective amount to subject and included according to right It is required that any one of 1-61 antibody or the composition of tau binding fragments, or (2) according to any one of claim 62-71's Composition.

128. suffer from or be easy to reduce in the subject with Alzheimer's or another tau lesions suffering from, suspecting The risk of AD or another tau lesions or the method for delay AD or another tau lesion breaking-outs, it, which includes applying to subject, controls (1) for treating effective dose is included according to any one of claim 1-61 antibody or the composition of tau binding fragments, or (2) root According to any one of claim 62-71 composition.

129. according to any one of claim 123-128 method, wherein the composition is applied by injecting.

130. according to any one of claim 123-129 method, wherein by the composition with 0.1mg/kg body weight extremely The antibody of 10mg/kg body weight or the dosage of binding fragment and between weekly and monthly, preferably fortnightly frequency is applied to institute Subject is stated, so as to treat the subject.

131. any one of claim 123-130 method, further comprise by least one type for being selected from the group Assess to monitor the therapeutic advance of subject：Mini mental status examination (Mini-Mental State Exam) (MMSE), Ah Er Cihai Mo's diseases assess scale-cognition (Alzheimer's Disease Assessment Scale-cognitive) (ADAS-COG) impression (Clinician Interview-Based Impression), based on clinician's talks (CIBI), neurology battery of tests (Neurological Test Battery) (NTB), dull-witted disability are assessed (Disability Assessment for Dementia) (DAD), clinical dementia grading-box summation (Clinical Dementia Rating-sum of boxes) (CDR-SOB), neuropsychopathy application form (Neuropsychiatric Inventory) (NPI), positron emission tomography (PET imagings) scanning (Positron Emission Tomography (PET Imaging) scan) and magnetic resonance imaging (MRI) scanning (Magnetic Resonance Imaging (MRI)scan)。

132. the method for claim 131, wherein the type of the assessment is neurology battery of tests (Neurological Test Battery)(NTB)。

133. the method for claim 131, wherein the type of the assessment is mini mental status examination (Mini-Mental State Exam)(MMSE)。

134. any one of claim 128-133 method, wherein the antibody or its tau binding fragment include SEQ ID NO.14 weight chain variable district and SEQ ID NO.26 light chain variable district.

135. any one of claim 128-133 method, wherein the antibody or its tau binding fragment include SEQ ID NO.16 weight chain variable district and SEQ ID NO.26 light chain variable district.

136. any one of claim 128-133 method, wherein the antibody or its tau binding fragment include SEQ ID NO.17 weight chain variable district and SEQ ID NO.26 light chain variable district.

137. any one of claim 128-133 method, wherein the antibody or its tau binding fragment include SEQ ID NO.25 weight chain variable district and SEQ ID NO.26 light chain variable district.

138. any one of claim 128-133 method, wherein the antibody or its tau binding fragment include SEQ ID NO.16 weight chain variable district and SEQ ID NO.27 light chain variable district.

139. any one of claim 128-133 method, wherein the antibody or its tau binding fragment include SEQ ID NO.17 weight chain variable district and SEQ ID NO.27 light chain variable district.

140. any one of claim 123-139 method, it further comprises controlling at least one others of effective dose Treat agent and be simultaneously or sequentially applied to the subject.

The method of 141. claims 140, wherein other therapeutic agents are selected from the group：Anti-apoptotic compound, metal-chelating Agent, DNA repair inhibitors, 3-APS (3APS), the disulfonic acid of 1,3- third (1,3PDS), secretion zymoexciter, β-point Secrete enzyme inhibitor, inhibitors of gamma-secretase, beta-amyloid peptide, tau- peptides, anti-amyloid beta antibody, neurotransmitter, β-lamella disrupting agent, anti-inflammatory molecular and anticholinesterase；And its any pharmaceutically acceptable salt.

The method of 142. claims 141, wherein the anticholinesterase be Tacrine, profit cut down the bright of this, donepezil, Galanthamine or nutritious supplementary pharmaceutical；And its any pharmaceutically acceptable salt.

The method of 143. claims 140, wherein other therapeutic agents are selected from beta-amyloid peptide (for example, N- ends Amyloid-beta peptide), it may or may not be conjugated with other compounds, such as the diphtheria toxin of mutation；For β-amylaceous egg White antibody, such as bapineuzumab, solaneuzumab, gantenerumab, crenezumab, ponezumab and IVIG Immunoglobulin, other immunotherapies of A beta oligomers are targetted, prevent the compound of tau hyperphosphorylations, and targeting tau aggregations Other actively and passively immunotherapies of body；And its any pharmaceutically acceptable salt.

The method of 144. claims 140, wherein other therapeutic agents be selected from amyloid-beta agglutination inhibitor (such as Tramiprosate), inhibitors of gamma-secretase (such as semagacestat) and gamma secretase modulators (tarenflurbil)；And its any pharmaceutically acceptable salt.

The method of 145. claims 140, wherein other therapeutic agents are selected from acetylcholinesteraseinhibitors inhibitors (for example, more Donepezil, profit cut down the bright of this, galanthamine, Tacrine, nutritious supplementary pharmaceutical), N-methyl-D-aspartate (NMDA) receptor antagonist Agent (such as Memantine), DNA repair inhibitors (for example, pirenzepine or its metabolin), transition metal chelator, growth because Son, hormone, nonsteroid anti-inflammatory drugs (NSAID), antioxidant, lipid lowering agent, selective phosphodiesterase inhibitors, tau aggregations Inhibitor, kinases inhibitor, resist mitochondria dysfunction Pharmacological inhibitors, neurotrophin, heat shock protein suppress Agent, lipoprotein associated phospholipase A₂Inhibitor, Memantine, anti-apoptotic compound, metal-chelator, DNA repair inhibitors, 3- ammonia Base -1- propane sulfonic acid (3APS), the disulfonic acid of 1,3- third (1,3PDS), secretion zymoexciter, beta-secretase inhibitor, gamma-secretase Inhibitor, beta-amyloid peptide, amyloid beta antibody, neurotransmitter, β-lamella disrupting agent, anti-inflammatory molecular；And its appoint What pharmaceutically acceptable salt.

The method of 146. claims 140, wherein other therapeutic agents are selected from BACE inhibitor；Muscarinic antagonist；Courage Alkali esterase inhibitor；Gamma-secretase inhibitors；Gamma secretase modulators；HMG-CoA reductase inhibitor；Nonsteroid anti-inflammatory Medicine；N-methyl-D-aspartate receptor antagonist；Anti-amyloid antibodies；Vitamin E；Nicotinic acetylcholine receptor swashs Dynamic agent；CB1 receptor inverse agonists or CB1 receptor antagonists；Antibiotic；Growth hormone cinogenic agent (secretagogue)；Group Amine H3 antagonists；AMPA activators；PDE4 inhibitor；GABA_AInverse agonists, amyloid aggregation inhibitor；Glycogensynthase Kinases beta inhibitor；The accelerator of α secretase activities；PDE-10 inhibitor, cholesterol absorption inhibitor, and its it is any pharmaceutically Acceptable salt.

The method of 147. claims 140, wherein other therapeutic agents are secondary antibodies.

The method of 148. claims 147, wherein the secondary antibody be selected from another tau antibody bapineuzumab, Solaneuzumab, gantenerumab, crenezumab, ponezumab and IVIG immunoglobulin.

149. assessments suffer from, suspect the side for suffering from or being easy to the subject with Alzheimer's or another tau lesions Method, methods described include the antibody or tau binding fragments and the biology from subject that test right requires any one of 1-61 The step of combination of the component of sample, wherein the Alzheimers in the detection instruction subject combined with biological sample Sick or another tau lesions.

The method of 150. claims 149, wherein the biological sample is biopsy, CSF, blood, serum or blood plasma sample Product.

Any one of 151. claim 118-121 host cell is producing and the people tau antibody combined or its tau bonding pad Purposes in the method for section, methods described include culture and cause the expression nucleic acid and produce the antibody or its tau is combined Fragment.

The antibody or binding fragment or (2) that 152. (1) include any one of claim 1-61 are appointed according in claim 62-71 The composition of one is used to suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect preparing Subject in treat purposes in the medicine of Alzheimer's or another tau lesions, or suffer from, suspect suffer from or It is easy to treat Alzheimer's or another tau in the subject with Alzheimer's or another tau lesions Purposes in the method for lesion.

The antibody or binding fragment or (2) that 153. (1) include any one of claim 1-61 are appointed according in claim 62-71 The composition of one prepare be used for promote tau aggregations from suffer from, suspects suffer from or be easy to suffer from Alzheimer's or Another purposes in the medicine removed in the brain of the subject of tau lesions, or promoting tau aggregations from suffering from, suspect trouble There is or is easy to the purposes in the method removed in the brain of the subject with Alzheimer's or another tau lesions.

The antibody or binding fragment or (2) that 154. (1) include any one of claim 1-61 are appointed according in claim 62-71 The composition of one is used to suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect preparing Subject in slow down AD or another tau lesions progress medicine in purposes, or suffer from suffering from, suspecting or be easy to suffer from In the method for having the progress for slowing down AD or another tau lesions in the subject of Alzheimer's or another tau lesions Purposes, including apply the antibody or bonding pad that (1) of therapeutically effective amount include any one of claim 1-61 to subject Section or (2) are according to any one of claim 62-71 composition.

The antibody or binding fragment or (2) that 155. (1) include any one of claim 1-61 are appointed according in claim 62-71 The composition of one is used to suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect preparing Subject in improve AD or another tau lesions symptom medicine in purposes, or suffer from suffering from, suspecting or be easy to suffer from In the method for having the symptom for improving AD or another tau lesions in the subject of Alzheimer's or another tau lesions Purposes.

The antibody or binding fragment or (2) that 156. (1) include any one of claim 1-61 are appointed according in claim 62-71 The composition of one prepare be used for treat, prevent or reverse suffer from, suspect suffer from or be easy to suffer from Alzheimer's or Another purposes in the medicine of cognition in the subject of tau lesions, or suffer from treatment, prevention or reverse, suspect and suffer from Or it is easy to the purposes in the method for the cognition in the subject with Alzheimer's or another tau lesions.

The antibody or binding fragment or (2) that 157. (1) include any one of claim 1-61 are appointed according in claim 62-71 The composition of one is used to suffer from or be easy to suffer from Alzheimer's or another tau lesions suffering from, suspect preparing Subject in reduce use in the risk of AD or another tau lesions or the medicine of delay AD or another tau lesions breaking-out On the way, or suffer from, suspect suffer from or be easy to reduce in the subject with Alzheimer's or another tau lesions AD or Another purposes in the risk of tau lesions or the method for delay AD or another tau lesion breaking-outs.

Any one of 158. claim 152-157 purposes, wherein the composition is applied by injecting.

Any one of 159. claim 150-158 purposes, wherein by the composition with 0.01mg/kg body weight to 100mg/ The antibody of kg body weight or the dosage of binding fragment and the frequency between weekly and monthly are applied to the subject, so as to treat The subject.

Any one of 160. claim 152-159 purposes, further comprise by least one type for being selected from the group Assess to monitor the therapeutic advance of subject：Mini mental status examination (Mini-Mental State Exam) (MMSE), Ah Er Cihai Mo's diseases assess scale-cognition (Alzheimer's Disease Assessment Scale-cognitive) (ADAS-COG) impression (Clinician Interview-Based Impression), based on clinician's talks (CIBI), neurology battery of tests (Neurological Test Battery) (NTB), dull-witted disability are assessed (Disability Assessment for Dementia) (DAD), clinical dementia grading-box summation (Clinical Dementia Rating-sum of boxes) (CDR-SOB), neuropsychopathy application form (Neuropsychiatric Inventory) (NPI), positron emission tomography (PET imagings) scanning (Positron Emission Tomography (PET Imaging) scan) and magnetic resonance imaging (MRI) scanning (Magnetic Resonance Imaging (MRI)scan)。

The purposes of 161. claims 160, wherein the type of the assessment is neurology battery of tests (NTB).

The purposes of 162. claims 160, wherein the type of the assessment is mini mental status examination (MMSE).

Any one of 163. claim 152-162 purposes, wherein the antibody or its tau binding fragment include SEQ ID NO.14 weight chain variable district and SEQ ID NO.26 light chain variable district.

Any one of 164. claim 152-162 purposes, wherein the antibody or its tau binding fragment include SEQ ID NO.16 weight chain variable district and SEQ ID NO.26 light chain variable district.

Any one of 165. claim 152-162 purposes, wherein the antibody or its tau binding fragment include SEQ ID NO.17 weight chain variable district and SEQ ID NO.26 light chain variable district.

Any one of 166. claim 152-162 purposes, wherein the antibody or its tau binding fragment include SEQ ID NO.25 weight chain variable district and SEQ ID NO.26 light chain variable district.

Any one of 167. claim 152-162 purposes, wherein the antibody or its tau binding fragment include SEQ ID NO.16 weight chain variable district and SEQ ID NO.27 light chain variable district.

Any one of 168. claim 152-162 purposes, wherein the antibody or its tau binding fragment include SEQ ID NO.17 weight chain variable district and SEQ ID NO.27 light chain variable district.

Any one of 169. claim 152-169 purposes, it further comprises controlling at least one others of effective dose Treat agent and be simultaneously or sequentially applied to the subject.

The purposes of 170. claims 169, wherein other therapeutic agents are selected from the group：Anti-apoptotic compound, metal-chelating Agent, DNA repair inhibitors, 3-APS (3APS), the disulfonic acid of 1,3- third (1,3PDS), secretion zymoexciter, β-point Secrete enzyme inhibitor, inhibitors of gamma-secretase, beta-amyloid peptide, anti-amyloid beta antibody, neurotransmitter, β-lamella Disrupting agent, anti-inflammatory molecular and anticholinesterase；And its any pharmaceutically acceptable salt.

The purposes of 171. claims 170, wherein the anticholinesterase be Tacrine, profit cut down the bright of this, donepezil, Galanthamine or nutritious supplementary pharmaceutical；And its any pharmaceutically acceptable salt.

The purposes of 172. claims 169, wherein other therapeutic agents are selected from beta-amyloid peptide (for example, N- ends Amyloid-beta peptide), it may or may not be conjugated with other compounds, such as the diphtheria toxin of mutation；For β-amylaceous Other anti-tau antibody of albumen, as bapineuzumab, solaneuzumab, gantenerumab, crenezumab, Ponezumab and IVIG immunoglobulins, other immunotherapies of A beta oligomers are targetted, prevent the chemical combination of tau hyperphosphorylations Thing, and other actively and passively immunotherapies of targeting tau aggregations；And its any pharmaceutically acceptable salt.

The purposes of 173. claims 169, wherein other therapeutic agents be selected from amyloid-beta agglutination inhibitor (such as Tramiprosate), inhibitors of gamma-secretase (such as semagacestat) and gamma secretase modulators (tarenflurbil)；And its any pharmaceutically acceptable salt.

The purposes of 174. claims 169, wherein other therapeutic agents are selected from acetylcholinesteraseinhibitors inhibitors (for example, more Donepezil, profit cut down the bright of this, galanthamine, Tacrine, nutritious supplementary pharmaceutical), N-methyl-D-aspartate (NMDA) receptor antagonist Agent (such as Memantine), DNA repair inhibitors (for example, pirenzepine or its metabolin), transition metal chelator, growth because Son, hormone, nonsteroid anti-inflammatory drugs (NSAID), antioxidant, lipid lowering agent, selective phosphodiesterase inhibitors, tau aggregations Inhibitor, kinases inhibitor, resist mitochondria dysfunction Pharmacological inhibitors, neurotrophin, heat shock protein suppress Agent, lipoprotein associated phospholipase A₂Inhibitor, Memantine, anti-apoptotic compound, metal-chelator, DNA repair inhibitors, 3- ammonia Base -1- propane sulfonic acid (3APS), the disulfonic acid of 1,3- third (1,3PDS), secretion zymoexciter, beta-secretase inhibitor, gamma-secretase Inhibitor, beta-amyloid peptide, amyloid beta antibody, neurotransmitter, β-lamella disrupting agent, anti-inflammatory molecular；And its appoint What pharmaceutically acceptable salt.

The purposes of 175. claims 167, wherein other therapeutic agents are selected from BACE inhibitor；Muscarinic antagonist；Courage Alkali esterase inhibitor；Gamma-secretase inhibitors；Gamma secretase modulators；HMG-CoA reductase inhibitor；Nonsteroid anti-inflammatory Medicine；N-methyl-D-aspartate receptor antagonist；Anti-amyloid antibodies；Vitamin E；Nicotinic acetylcholine receptor swashs Dynamic agent；CB1 receptor inverse agonists or CB1 receptor antagonists；Antibiotic；Growth hormone cinogenic agent；Histamine H 3 antagonists；AMPA Activator；PDE4 inhibitor；GABA_AInverse agonists, amyloid aggregation inhibitor；Glycogen synthase kinase beta inhibitor；α points Secrete the accelerator of enzymatic activity；PDE-10 inhibitor, cholesterol absorption inhibitor, and its any pharmaceutically acceptable salt.

The purposes of 176. claims 169, wherein other therapeutic agents are secondary antibodies.

The purposes of 177. claims 169, wherein the secondary antibody be selected from another tau antibody bapineuzumab, Solaneuzumab, gantenerumab, crenezumab, ponezumab and IVIG immunoglobulin.

The composition of 178. antibody comprising any one of claim 1-61 or binding fragment is suffered from for assessment, cherished preparing The purposes in the medicine for suffering from or being easy to the subject with Alzheimer's or another tau lesions is doubted, or is being commented Estimate and suffer from, suspect the purposes for suffering from or being easy to suffer from the method for the subject of Alzheimer's or another tau lesions, The assessment includes the antibody or tau binding fragments and the biological sample from subject that test right requires any one of 1-61 Component combination the step of, wherein the detection combined with biological sample indicates the Alzheimers in the subject Sick or another tau lesions.

The purposes of 179. claims 178, wherein the biological sample is biopsy, CSF, blood, serum or blood plasma sample Product.